Means and methods for treating herpesvirus infection

ABSTRACT

The present invention provides herpesviruses, such as EBV, which lack at least one viral miRNA. Such herpesviruses lacking at least one viral miRNA are advantageously not capable of packaging their genome into the capsid, thereby producing HVLPs, which are substantially free of their herpesvirus genome or the nucleic acid molecule encoding the proteinaceous part of the HVLP and viral miRNA. Such HVLPs may be used as vaccine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/080,292, filed Aug. 27, 2018, now U.S. Pat. No. 11,602,560, which isa U.S. national phase application of International PCT PatentApplication No. PCT/EP2017/054615, which was filed on Feb. 28, 2017,which claims priority to Luxembourg Application No. 93002, filed Mar.17, 2016, and European Application No. 16000493.3, filed Mar. 1, 2016.These applications are incorporated herein by reference in theirentireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing XML associated with this application is provided inXML file format and is hereby incorporated by reference into thespecification. The name of the XML file containing the Sequence ListingXML is SCHI_008_01 US_ST26.xml. The XML file is 31,984 bytes, andcreated on Mar. 10, 2023, and is being submitted electronically viaUSPTO Patent Center.

Epstein-Barr virus (EBV) is an oncogenic herpes virus that infects morethan 90% of the human population worldwide but causes an enormous threatespecially to the immune-compromised host. EBV is responsible for anumber of acute and chronic, inflammatory, autoimmune and malignantdisorders, which include several types of severe and life-threateninglymphoproliferative diseases in immunosuppressed patients. Patients atrisk are candidates for solid organ or hematopoietic stem celltransplantation (SOT/SCT) as well as patients with HIV infection, andpatients with congenital immunodeficiency. An important risk factor forEBV-associated PTLD is seronegativity at the time of transplant, whichexplains particularly high rates of PTLD in children. Depending on thetype of transplant, up to 15% of pediatric transplant patients areaffected, and eventually 20% of those succumb to the PTLD (Mynarek etal., Clin Dev Immunol. 2013; 2013:814973).

EBV is both ubiquitous and immunogenic. This oncogenic herpes virus(IARC Working Group, IARC Monogr. Eval. Carcinog. Risks Hum. 94, 46-70(2010)) has evolved multiple genes to fend off immune responses when itsinfection is established (Ressing et al., Semin. Cancer Biol. 18,397-408 (2008)). However, these viral genes do not accumulateimmediately on infection of B-lymphocytes, EBV's primary target cells.Thus, early infection should be its Achilles heel, a window when EBV isunprotected from the host's immune response.

A prophylactic vaccine is thought to be the most effective step towardsreducing the burden of EBV-associated malignant and non-malignantdiseases. Not only PTLD but also infectious mononucleosis (IM) inchildren and adolescents and endemic Burkitt lymphoma are diseases thatwere identified as indications of a prophylactic EBV vaccine (Balfour,Curr Opin Virol., 2014, vol.6, pp. 1-5; Cohen et al., Sci Transl Med.,2011, 3(107):107fs7; Cohen et al., Vaccine, 2013, 31 Suppl 2:6194-6).These diseases are also secondary targets for efficacy trials with theplanned VLP vaccine. Epstein made the first vaccine proposal almost 40years ago, but a viable vaccine is still not available. Progress issignificantly hampered by the lack of tractable animal models exceptsubhuman primates and the complexity of the virus. Most vaccine effortsto prevent EBV infection or related diseases have focused on gp350,which is the most abundant glycoprotein of the virion and the principaltarget of naturally occurring neutralizing antibodies. Vaccination witha soluble form of gp350 reduced the rate of IM in EBV seronegativeadults, but had no effect on the rate of EBV infection (Sokal et al., JInfect Dis., 2007, 196(12):1749-53). Another gp350-based vaccine inducedantibody responses in EBV- negative children with chronic kidney diseaseawaiting transplantation, but did not prevent post-transplant adverseconsequences of EBV-associated diseases (Rees et al., Transplantation,2009, 88(8):1025-9). In sum, the few clinical vaccination trialsindicate that a prophylactic vaccination against EBV-associated diseasesis feasible, but the trials also document that current vaccinationstrategies need to be considerably improved to prevent primary infectionand/or EBV-associated diseases in all vaccinees. Thus, there is anurgent unmet need of a vaccine against EBV.

The present inventors revealed that EBV on infecting primary B-cellsefficiently suppresses multiple arms of adaptive immune responses withits encoded miRNAs. They control all three signals required forantigen-specific T-cell activation and recognition: (i) processing andpresentation of antigenic peptides to T-cells; (ii) levels of importantco-receptors on EBV-infected B-cells that modulate T-cell activation;and (iii) secretion of pro-inflammatory and other cytokines thatpolarize naive CD4⁺ T-cells to antiviral Th1 helper cells, therebymiRNAs protect newly infected B-lymphocytes from immune eradication,allowing EBV's life-long success. Namely, the present inventors foundthat EBV's miRNAs target cellular genes directly to inhibit secretion ofcytokines, antigen processing, recognition of virus-infected cells byEBV-specific CD4⁺ and CD8⁺ T-cells, and/or differentiation of naïveT-cells to antiviral Th1 cells. The variety and the massive inhibitionof adaptive immune responses by multiple miRNAs of a single pathogen wasunexpected and unprecedented.

The results obtained by the present inventors can also explain theabundance of miRNAs in complex persisting viruses, and clarify how ahuman pathogen can evade elimination for the lifetime of its host inspite of intense adaptive immune responses. This global suppressionallows the virus to express antigenic functions in cells neededinitially to establish its life-long infection evading destruction byT-cells.

By using a human cellular model closely mimicking natural infection thepresent inventors found that EBV's miRNAs counteract multiple pathwaysof antiviral adaptive immunity as described in more detail in theappended Examples and illustrated in the Figures.

Given the surprising findings of the present inventors on the prominentrole of EBV miRNAs in infection, it would be desirous to eliminate suchmiRNAs not only from EBV, but also from other herpes viruses to whichEBV belongs. In fact, it is known that, apart from EBV, also otherherpesviruses have miRNAs which are envisioned to have such prominentrole as the miRNAs of EBV as well (Boss et al,. 2009, Trends inMicrobiology, vol. 17, issue 12, pp. 544-553).

Accordingly, the present invention provides herpesviruses, such as gammaherpes viruses, e.g. EBV, that lack at least one viral miRNA orpreferably all viral miRNAs. Such herpesviruses that lack at least oneviral miRNA or preferably all viral miRNAs are advantageously also notcapable of packaging their genome or the nucleic acid molecule encodingthe proteinaceous part of the virus into the capsid, thereby producingherpesvirus-like particles (HVLPs) or Epstein-Barr virus-like particles(EBVLP) which are substantially free of their herpesvirus genome or thenucleic acid molecule encoding the proteinaceous part of the virus andmiRNA.

Virus-like particles (VLPs) are structural similar to mature virions butlack the viral genome. Therefore, VLPs are promising candidates forvaccination. Accordingly, such HVLPs end EBVLPs of the present inventionmay be used as herpesvirus vaccines.

Therefore, the present invention provides a Herpes virus-like particle(HVLP) comprising Herpes viral proteins which are encoded by at leastone nucleic acid molecule which still comprises miRNA coding lociencoding Herpes viral miRNAs, wherein at least one of said miRNA codingloci is genetically modified.

The term “Herpes virus-like particle” and “HVLP” are usedinterchangeably herein and relate to particles, which sharemorphological and immunological properties with infectious Herpes virusparticles, but lack the viral genome and thus are preferably not capableof propagating infection and/or replicating in a suitable host cell.HVLPs in accordance with the present invention can in principle compriseall Herpes viral proteins of the wild type virus and thus preferablyhave a typical Herpes virus structure as can be analyzed by electronmicroscopy, i.e. they have a capsid, a tegument and an outer membrane.Thus, a HVLP of the present invention may comprise Herpes viral capsidor capsid precursor proteins, surface proteins, envelope proteins, coatproteins, shell proteins, glycoproteins, tegument proteins, proteinsgiving rise to B-cell and/or T-cell epitopes. However, certain Herpesvirus proteins of the HVLP of the present invention may be geneticallymodified compared to the wild type virus strain, as described herein. Itis however also envisaged that the HVLP lacks one or more non-essentialviral proteins. Such a non-essential viral protein is incorporated inthe wild type HVLP but is not essential for the formation of the HVLP,as can be detected by electron microscopy of a HVLP produced accordingto the methods described herein in absence of the polypeptide encoded bysaid gene. A HVLP of the present invention preferably comprises orconsists of proteins originating from one Herpes virus (e.g.Epstein-Barr virus) and even more preferred from one Herpes virusstrain, e.g. Epstein-Barr virus strain B95.8, Epstein-Barr virus type 1or Epstein-Barr virus type 2, Epstein-Barr virus strain B95.8 beingpreferred.

The term “Herpes viral proteins” as used herein comprises proteins ofwild type Herpes virus strains, but also proteins that are not identicalto proteins of wild type Herpes virus strains as regards the sequence,but share at least 99%, at least 98%, at least 97%, at least 96%, atleast 95%, at least 94%, at least 93%, at least 92%, at least 91%, atleast 90%, at least 85%, at least 80%, or at least 75% sequence homologywith proteins of wild type Herpes virus strains. By way of example, wildtype Epstein-Barr virus proteins are preferably encoded by theprototypic Epstein-Barr virus B95.8 (Genbank Accession number V01555).Moreover, wild type Epstein-Barr virus proteins can be encoded byEpstein-Barr virus type 1 (Genbank Accession number NC_007605.1), or byEpstein-Barr virus type 2 (Genbank Accession number NC_009334.1).

“Sequence identity” or “sequence homology” refers to the percentage ofresidue matches between at least two polypeptide or polynucleotidesequences aligned using a standardized algorithm. Such an algorithm mayinsert, in a standardized and reproducible way, gaps in the sequencesbeing compared in order to optimize alignment between two sequences, andtherefore achieve a more meaningful comparison of the two sequences. Forpurposes of the present invention, the sequence identity between twoamino acid sequences is determined using the NCBI BLAST program version2.3.0 (Jan. 13, 2016) (Altschul et al., Nucleic Acids Res. (1997)25:3389-3402). Sequence identity of two amino acid sequences can bedetermined with blastp set at the following parameters: Matrix:BLOSUM62, Word Size: 3; Expect value: 10; Gap cost: Existence=11,Extension=1; Compositional adjustments: Conditional compositional scorematrix adjustment.

A HVLP of the present invention may further comprise in addition toHerpes virus proteins one or more artificial proteins. The term“artificial proteins” as used herein relates to proteins, which are notencoded by the wild type Herpes virus. Artificial proteins may beselected from the group of additional foreign antigenic sequences,cytokines, CpG motifs, g-CMSF, fluorescent proteins, proteins useful forpurification purposes of the particles or for attaching a label,proteinaceous structures required for transport processes and others.

The HVLP of the present invention relates to a particle whoseproteinaceous part is encoded by the at least one nucleic acid molecule.Accordingly, the at least on nucleic acid molecule may comprise allgenes of the wild type Herpes virus and thus encode all Herpes viralproteins of the wild type Herpes virus and may further comprise also allnon-coding nucleic acid sequences (e.g. miRNA coding loci, cis-actingelements) of the Herpes virus. Thus, the at least one nucleic acidmolecule may comprise all coding and non-coding nucleic acid sequencesof the wild type virus. However, certain genes, coding sequences ornon-coding sequences of the at least one nucleic acid molecule may bemodified compared to the wild type Herpes virus, as described herein.Furthermore, the at least one nucleic acid molecule may lack one or moregenes compared to the wild type virus which are not essential for theformation of a HVLP, as can be determined by electron microscopy of aHVLP produced according to the methods described herein in absence ofthe polypeptide encoded by said gene. The at least one nucleic acidmolecule is thus capable of conferring the production of the HVLPs ofthe present invention in a suitable host cell. By way of example, the atleast one nucleic acid molecule which encodes an Epstein-Barr virus-likeparticle (EBVLP) may comprise all coding and/or non-coding sequences ofwild type Epstein-Barr virus (e.g. EBV strain B95.8), which may compriseone or more genetic modifications such as (i) functional inactivation ofone or more viral oncogenes required for B-cell transformation (e.g.EBNA1, EBNA-LP, EBNA2, LMP1, LMP2, EBNA3A, and EBNA3C), (ii) functionalinactivation of one or more cis-acting elements (e.g. terminal repeats,TR) or viral genes encoding portal proteins (e.g. BFLF1, BBRF1, BGRF1,BDRF1, BALF3, BFRF1A, and BFRF1) which are essential for cleavage andpackaging of the at least one nucleic acid molecule, (iii) functionalinactivation of one or more viral genes required for inducing virussynthesis (e.g. BZLF1, BRLF1 and BMLF1), and/or (iv) functionalinactivation of at least one miRNA coding loci. Thus, in a preferredembodiment of the invention the at least one nucleic acid molecule isone nucleic acid molecule that differs from a wild type EBV genome onlywith respect to the above identified features. It is however alsoenvisaged that the EBVLP lacks one or more non-essential viral proteins.Such a non-essential viral protein is incorporated in the wild typeEBVLP but is not essential for the formation of the EBVLP, as can bedetected by electron microscopy of an EBVLP produced according to themethods described herein in absence of the polypeptide encoded by saidgene. Furthermore, the at least one nucleic acid molecule preferablycomprises or consists of nucleic acid sequences originating from oneHerpes virus (e.g. Epstein-Barr virus) and even more preferred from oneHerpes virus strain, e.g. Epstein-Barr virus strain B95.8, Epstein-Barrvirus type 1 or Epstein-Barr virus type 2, Epstein-Barr virus strainB95.8 being preferred. In a further preferred embodiment of theinvention the at least one nucleic acid molecule which encodes the EBVLPdoes not encode a functional BHRF1 protein.

The terms “Herpes virus genes” and “wild type Herpes virus genes” areused interchangeably herein, comprise genes of wild type Herpes virusstrains, but also genes that are not identical to genes of wild typeHerpes virus strains as regards the sequence, but share at least 99%, atleast 98%, at least 97%, at least 96%, at least 95%, at least 94%, atleast 93%, at least 92%, at least 91%, at least 90%, at least 85%, atleast 80%, or at least 75% sequence homology with genes of wild typeHerpes virus strains. By way of example, wild type Epstein-Barr virusgenes are preferably encoded by the prototypic Epstein-Barr virus B95.8.Moreover, wild type Epstein-Barr virus type 1 genes are encoded byEpstein-Barr virus type 1 (Genbank Accession number NC_007605.1), wildtype Epstein-Barr virus type 2 genes are encoded by Epstein-Barr virustype 2 (Genbank Accession number NC_009334.1).

Thus, upon expression of the at least one nucleic acid in a suitablehost cell the Herpes viral proteins are expressed, resulting in theformation of HVLPs. Consequently, the HVLP of the present invention isobtainable by a host cell which comprises the at least one nucleic acidencoding the Herpes viral proteins which still comprises miRNA codingloci encoding Herpes viral miRNAs, wherein at least one of said miRNAcoding loci is genetically modified. However, the HVLP of the presentinvention is after formation released from the host cells, preferablyvia the endosomal sorting complex required for transport (ESCRT). Thus,a HVLP of the present invention further comprises cellular componentsderived from the host cell, such as lipids, proteins, glycoproteins(e.g. CD63), nucleic acids (e.g. mRNAs and miRNAs), cell membranes andothers.

The term “cell membranes” as used herein relates to lipids thatnaturally form cell membranes by spontaneously arranging to form a lipidbilayer, such as amphiphatic phospholipids, wherein after self- assemblythe hydrophobic regions of the amphiphatic phospholipids form the innerpart of the bilayer whereas the hydrophobic regions form the outer faceof the membrane. Preferably, such cell membranes originate from the hostcell from which a HVLP of the present invention originates and forms theouter membrane of the HVLP of the present invention. Furthermore, such acell membrane comprises proteins, e.g. EBV structural proteins such asgp350 and/or LMP-1 in case of an EBVLP.

The term “herpesvirus” as used herein relates to any virus of the familyof Herpesviridae. However, preferred are herpesviruses which infecthumans such as Human herpesvirus 1 (Herpes simplex virus 1 or HSV-1),Human herpesvirus 2 (Herpes simplex virus 2 or HSV-2), Human herpesvirus3 (Varicella-zoster virus or VZV), Human herpesvirus 4 (Epstein-Barrvirus or EBV), Human herpesvirus 5 (Human cytomegalovirus or HCMV),Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8 (Kaposi'ssarcoma-associated herpesvirus or KSHV). Even more preferred is theHuman herpesvirus 4 (Epstein-Barr virus or EBV), which relates to EBVtype 1 and EBV type 2 and preferably EBV strain B95.8. However, alsoenvisioned are Murid herpesvirus 4 (e.g. Murine gammaherpesvirus 68 orMHV-68) and Bovine herpesvirus 1 (e.g. Infectious bovine rhinotracheitisvirus).

The terms “protein” or “polypeptide” are used interchangeably herein andrefer to a molecule comprising a polymer of amino acids linked togetherby peptide bonds. Said term is not meant herein to refer to a specificlength of the molecule. A polypeptide comprises an amino acid sequence,and, thus, sometimes a polypeptide comprising an amino acid sequence isreferred to herein as a “polypeptide comprising a polypeptide sequence”.Thus, herein the term “polypeptide sequence” is interchangeably usedwith the term “amino acid sequence”.

The term “amino acid” or “aa” refers to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

The terms “polynucleotide”, “nucleotide sequence” “nucleic acidmolecule” or “nucleic acid” are used interchangeably herein and refer toa polymeric form of nucleotides, which are usually linked from onedeoxyribose or ribose to another. The term “polynucleotide” preferablyincludes single and double stranded forms of DNA. A nucleic acidmolecule may include both sense and antisense strands of RNA (containingribonucleotides), cDNA, genomic DNA, and synthetic forms and mixedpolymers of the above.

The Herpes viral proteins of the HVLP of the invention are encoded by atleast one nucleic acid molecule. Thus, the Herpes viral proteins of theHVLP may be encoded by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or more nucleic acid molecules. However, in a preferred embodiment ofthe invention the Herpes viral proteins of the HVLP are encoded by onenucleic acid molecule.

The at least one nucleic acid molecule encoding the Herpes viralproteins still comprises miRNA coding loci encoding Herpes viral miRNAsof the HVLP, wherein at least one of said miRNA coding loci isgenetically modified. Such miRNAs coding loci are derived from theHerpes virus and therefore encode viral miRNAs. The term “stillcomprises” in this context thus means that the miRNA coding locioriginate from a Herpes virus, preferably from the same Herpes virusfrom which the proteins originate, and are still comprised by the atleast one nucleic acid molecule encoding the herpes viral proteins. Orin other words, the at least one nucleic acid molecule comprises Herpesviral coding and non-coding nucleic acid sequences including the miRNAcoding loci. Such viral miRNAs are usually expressed in the host celland thus packaged in the viral particle upon virus synthesis.Consequently, the viral miRNAs are as well packaged in the HVLPs uponproduction of the HVLPs as described herein. However, such viral miRNAsmay counteract the antiviral immunity of the host, which may bedetrimental upon vaccination with a HVLP. Thus, at least one miRNAcoding loci of the at least one nucleic acid molecule, which encodes atleast one miRNA, is genetically modified. However, also more than onemiRNA coding loci may be genetically modified, such as 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, or more. In a preferred embodiment of theinvention all viral miRNA coding loci are genetically modified. In afurther embodiment at least one miRNA coding loci of each miRNA clusterof the Herpes virus is genetically modified (e.g. at least one miRNAcoding loci of the BART cluster and at least one miRNA coding loci ofthe BHRF1 cluster in case of Epstein-Barr virus).

The term “miRNA” as used herein relates to small noncodingsingle-stranded RNAs of about 21 to 25 nucleotides in length. The5′-ends of miRNAs, the so-called seed sequences, recognize partiallycomplementary mRNA targets usually within their 3′ untranslated regionsand repress translational of these mRNAs. miRNA coding loci are firsttranscribed into longer primary miRNAs (pri-miRNAs) usually by RNApolymerase II. The RNase III enzyme Drosha then recognizes and cleavesthe pri-miRNAs to liberate hairpin structures, usually 60 to 80 nt long,called pre-miRNAs, which are transported into cytoplasm and furtherprocessed by another RNase III enzyme named Dicer to produce RNAduplexes. The RNA duplexes associate with Argonaute (Ago) proteins,Dicer, and GW182 in RNA-induced silencing complexes (RISC), where theyare unwound. Often, at this stage, one strand (the “star strand”) isdegraded, while the other strand (mature miRNA) is retained. The RISC isguided by the miRNAs to specifically recognize and regulate targetmRNAs. Thus, miRNAs are key regulators of a number of biologicalprocesses including developmental timing, differentiation and pattering,but also cellular proliferation, cell death, immune response,haematopoiesis, and cellular transformation or oncogenesis. One singlemiRNA may directly regulate the expression of hundreds of differentmRNAs.

By way of example, Herpes viral miRNAs are H1 to H8 and H11 to H18 ofHerpes simplex virus 1, H2 to H7 and H9 to H13 and H19 to H25 of Herpessimplex virus 2, miR-UL36-5p, miR-UL36-3p, miR-UL112, miR-US4,miR-US5-1, miR-US5-2, miR-US25-1-5p, miR-US25-1-3p, miR-US25-2-5p,miR-US25-2-3p, miR-US33-3p, miR-UL22A-5p, miR-UL22A-3p, miR-UL70-5p,miR-US33-5p, miR-UL70-3p and miR-UL112-1 of Cytomegalovirus, miR-K1-5p,miR-K2-5p, miR-K3-5p, miR-K3_+1_5, miR-K4-3p, miR-K4-5p, miR-K5-3p,miR-K6-3p, miR-K6-5p, miR-K7-3p, miR-K7-5p, miR-K8-3p, miR-K8-5p,miR-K9-3p, miR-K9-5p, miR-K10a (−3p), miR-K10b-(-3p),miR-K10b_+1_5(-3p), miR-K10b_+1_5(-3p), miR-K11-3p, miR-K12-3p,miR-K12-5p of Kaposi's sarcoma associated virus and Epstein-Barr viruspre-miRNAs miR-BHRF1-1, miR-BHRF1-2, miR-BHRF1-3, miR-BART1, miR-BART2,miR-BART3, miR-BART4, miR-BART5, miR-BART6, miR-BART7, miR-BART8,miR-BART9, miR-BART10, miR-BART11, miR-BART12, miR-BART13, miR-BART14,miR-BART15, miR-BART16, miR-BART17, miR-BART18, miR-BART19, miR-BART20,miR-BART21, miR-BART22, giving rise to four mature BHRF1 miRNAs and 40BART miRNAs. However, for the Herpes viruses varicella-zoster virus andHHV-7 no miRNAs have been identified so far. Thus, preferredherpesviruses of the invention are Human herpesvirus 1 (Herpes simplexvirus 1 or HSV-1), Human herpesvirus 2 (Herpes simplex virus 2 orHSV-2), Human herpesvirus 4 (Epstein-Barr virus or EBV), Humanherpesvirus 5 (Human cytomegalovirus or HCMV), Human herpesvirus 6,Human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus or KSHV).Even more preferred is the Human herpesvirus 4 (Epstein-Barr virus orEBV), which relates to EBV type 1 and EBV type 2 and preferably EBVstrain B95.8. In a further embodiment the present invention does notrelate to HVLPs derived from Herpes viruses that do not express miRNAs.

The term “genetically modified” with respect to miRNA coding loci, asused herein generally relates to any genetic modification or mutation,which causes a functional inactivation of the viral miRNA, such that theviral miRNA is no longer capable of interfering with translation of thetargeted mRNAs. In a preferred embodiment of the invention thenucleotide sequence of the miRNA coding loci is scrambled, such thatprecursors of the viral miRNAs are transcribed, but not processedfurther to give rise to functional miRNAs. Without being bound bytheory, such a scrambled miRNA coding sequence abrogate expression ofmature miRNAs in host cells and thus result in HVLPs that do not containviral miRNAs or their functional precursors. Such scrambled miRNA codingloci preferably result in primary miRNAs (pri-miRNAs) that have a wrong3D structure.

The term “wrong 3D structure” as used herein relates to a geneticallymodified pri-miRNA which is unable to fold into the specific hairpinstructures of pri-miRNAs and therefore is not processed to a maturemiRNA by the nuclear RNase III enzyme Drosha. However, the miRNA codingloci may also be deleted. Consequently, genetic modifications as usedherein effect that at least one Herpes viral miRNA is not expressed oronly partially expressed, at least one Herpes viral miRNA does not bindto its target sequence, at least one Herpes viral miRNA, pri-miRNA or aprecursor has a wrong 3D structure, at least one precursor of a Herpesviral miRNA is not further processed, at least one Herpes viral miRNA orits precursor are degraded by the cell, at least one Herpes viral miRNAcoding loci has a scrambled sequence, at least one Herpes viral miRNAcoding loci is deleted, and/or at least one Herpes viral miRNA or itsprecursor comprises mutations, deletions or insertions. In a preferredembodiment, genetic modifications as used herein effect that at leastone Herpes viral miRNA is not expressed or only partially expressed, atleast one Herpes viral miRNA does not bind to its target sequence, atleast one Herpes viral miRNA, pri-miRNA or a precursor has a wrong 3Dstructure, at least one precursor of a Herpes viral miRNA is not furtherprocessed, at least one Herpes viral miRNA or its precursor are degradedby the cell, at least one Herpes viral miRNA coding loci has a scrambledsequence, and/or at least one Herpes viral miRNA or its precursorcomprises mutations or insertions, but no Herpes viral miRNA coding lociis deleted or comprises deletions. Preferably, such geneticmodifications do not alter the wild type nucleotide composition and thegenomic architecture of the virus. A genetically modified miRNA codingloci can be identified by comparing the nucleotide sequence of saidgenetically modified miRNA coding loci with the nucleotide sequence ofthe corresponding miRNA coding loci of the wild type virus, i.e. in caseof EBV with the EBV reference strain with Genbank Accession numberAJ507799. Thus, a genetic modification of a miRNA coding loci of thepresent invention causes a deviation from the sequence of thecorresponding loci of the wild type virus (e.g. EBV strain AJ507799) andeffects that the miRNA is not expressed or only partially expressed, themiRNA does not bind to its target sequence, the miRNA or its precursorhas a wrong 3D structure, the miRNA is not further processed, the miRNAor its precursor are degraded by the cell, the miRNA coding loci has ascrambled sequence, the miRNA coding loci is deleted, and/or at leastone Herpes viral miRNA or its precursor comprises mutations, deletionsor insertions.

The present inventors surprisingly found that miRNAs of Epstein-Barrvirus encode functions that are immunosuppressive and repress adaptiveimmunity responses of the host. Accordingly, the genetic modification ofat least one of the miRNA coding loci, comprised by the at least onnucleic acid molecule, leads to an increased immune response whencompared to a HVLP comprising Herpes viral proteins which are encoded byat least one nucleic acid that comprises no genetically modified Herpesviral miRNA coding loci, wherein said increase is at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 365, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, 125%,150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%,900%, 1000% or more or preferably at least 5% as determined in the assayas described herein.

The term “assay” as described herein with respect to detection of anincreased immune response relates in general to any assay, which issuitable to detect an immune response, preferably of lymphocytes, suchas B-cells, T-cells, NKT-cells. By way of example the new synthesis orrelease of proinflammatory cytokines by immune cells, preferablylymphocytes, such as B-cells, T-cells or any other lymphocyte can bemeasured, by using a suitable immunological assay, such as aquantitative ELISA (Enzyme-linked Immunosorbent Assay). Such aquantitative ELISA can be used in context with the present invention tomeasure the cytokine concentration in the supernatant of immune cells,preferably lymphocytes, such as B-cells, T-cells or any other lymphocyteand more preferably B-cells. Cytokines that can be measured inaccordance with the present invention are in general all proinflammatorycytokines, e.g. IL-6 and TNF-α and other cytokines, e.g. IL-12(comprising proteins p35 and p40 encoded by the genes IL12A and IL12B,respectively), IL-12B (comprising two p40 proteins encoded by the gene11_12B) and IL-23 (comprising proteins p40 and p19 encoded by the genesIL12B and IL23A, respectively). In case of IL-12 it is known thatseveral EBV miRNAs inhibit the transcript of the gene IL12B and thusreduce the secretion of cytokines IL12, IL12B and IL23. Accordingly, theincreased immune response is preferably measured by measuring thecytokine concentration, using a quantitative ELISA, in the supernatantof B-cells (e.g. IL-12, IL-6, TNF-α) which have been incubated withHVLPs or EBVLPs that lack at least one Herpes virus or EBV miRNA inaccordance with the present invention (i.e. the Herpes viral proteins orEBV proteins of the HVLP or EBVLP are encoded by at least one nucleicacid molecule which still comprises miRNA coding loci encoding Herpesviral or EBV miRNAs, wherein at least one of said miRNA coding loci isgenetically modified) and compare the measured cytokine concentration tothe cytokine concentration measured in the supernatant of B-cells whichhave been incubated with HVLPs or EBVLPs that do not lack at least oneHerpes virus or EBV miRNA, wherein the HVLPs or EBVLPs that do not lackat least one Herpes virus or EBV miRNA are preferably encoded by anucleic acid molecule comprising miRNA coding loci, which are identicalto the wild type virus (i.e. reference strain AJ507799 in case of EBV).The B-cells in the described assay can be incubated for at least 3 h, atleast 6 h, at least 9 h, at least 12 h, at least 15 h, at least 18 h, atleast 21 h, at least 24 h, at least 27 h, at least 30 h, at least 36 hat least 39 h, at least 42 h, at least 45 h, at least 48 h, at least 54h, at least 60 h, at least 66 h, at least 72 h, at least 84 h, at least96 h or more and preferably 24 h or 36 h or more preferably anyintermediate between 24h to 36h. Furthermore, the new synthesis ofcytokines can be measured using quantitative RT-PCR with primersspecific for the cytokine transcript (IL-6, TNF-α, IL-12 or othercytokine transcripts). Thus, the increased immune response can bemeasured for example by measuring the IL-12 transcript of B-cells whichhave been incubated with HVLPs or EBVLPs as described herein by usingquantitative RT-PCR with IL-12 and preferably IL-12B transcript specificprimers and by comparing the results obtained from B-cells which havebeen incubated with HVLPs or EBVLPs that lack at least one Herpes virusor EBV miRNA in accordance with the present invention and from B-cellswhich have been incubated with HVLPs or EBVLPs that do not lack at leastone Herpes virus or EBV miRNA, wherein said HVLPs or EBVLPs that do notlack at least one Herpes virus or EBV miRNA are preferably encoded by anucleic acid molecule comprising miRNA coding loci, which are identicalto the wild type virus (i.e. reference strain AJ507799 in case of EBV).Moreover, the assay can be an assay, which uses immune cells, preferablylymphocytes, such as T-cells, NKT-cells, or other lymphocytes as aread-out to measure an increased immune response. Such immune cells,termed effector cells, recognize the B-cells incubated with HVLPs orEBVLs (i.e. B-cells that present antigenic epitopes derived from HVLPsor EBVLs). After epitope recognition, the immune cells respond with anincreased secretion of cytokines or even kill the B-cells incubated withHVLPs or EBVLPs. Thus, the increased immune response can be measured forexample by measuring the cytokine concentration in the supernatant ofimmune effector cells (e.g. GM-CSF or IFN-gamma), which have beenincubated with B-cells incubated with HVLPs or EBVLPs that lack at leastone Herpes virus or EBV miRNA compared to the cytokine concentration inthe supernatant of immune effector cells, which have been incubated withB-cells incubated with HVLPs or EBVLPs that do not lack at least oneHerpes virus or EBV miRNA, wherein said HVLPs or EBVLPs that do not lackat least one Herpes virus or EBV miRNA are preferably encoded by anucleic acid molecule comprising miRNA coding loci, which are identicalto the wild type virus (i.e. reference strain AJ507799 in case of EBV).The increased immune response can further be measured by measuring therelease of cytokines by effector cells by staining their surface forcytokines being secreted. The increased immune response of the effectorcells can also be measured by measuring the killing of the B-cellsincubated with HVLPs or EBVLs. Prior to the killing experiment, B-cellsincubated with HVLPs or EBVLPs that lack at least on viral miRNA andB-cells incubated with HVLPs or EBVLPs that do not lack at least onviral miRNA are stained with a dye (e.g. Calcein-acetoxymethlester).Calcein is released to the supernatant upon effector cell-mediatedkilling, where its concentration is proportional to the number of killedcells and can be quantified by fluorometric measurement. Thus, anincrease of Calcein is indicative of an increased immune response.Consequently, an increased immune response can be measured by comparingthe Calcein concentration in the supernatant of B-cells, incubated withHVLPs or EBVLPs that lack at least on viral miRNA and B-cells incubatedwith HVLPs or EBVLPs that do not lack at least on viral miRNA, whereinsaid HVLPs or EBVLPs that do not lack at least one Herpes virus or EBVmiRNA are preferably encoded by a nucleic acid molecule comprising miRNAcoding loci, which are identical to the wild type virus (i.e. referencestrain AJ507799 in case of EBV).

The term “increased immune response”, as used herein relates to anincreased immune response upon administration of HVLPs of the presentinvention to a subject or in an in vitro assay. Such an increase becomesevident when comparing the immune response caused by the HVLP of thepresent invention with the immune response caused by a HVLP comprisingHerpes viral proteins, which are encoded by at least one nucleic acidwhich does not comprise miRNA coding loci encoding Herpes viral miRNAsthat are genetically modified. Such an increased immune response in asubject is preferably an increased adaptive immune response such as ahumoral or a cellular immune response, i.e. a B-cell response or T-cellresponse. More preferably said increased immune response is an increasedT-cell response, such as a CD8+ or CD4+ T-cell response. Even morepreferred is an increased CD8+ T-cell response. Most preferably, saidincreased immune response is an increased B-cell, CD4+ T-cell and CD8+T-cell response. In case of EBVLPs such a CD8+ T-cell response issurprising as EBVLPs are inactivated vaccines, which are known not toinduce a CD8+ T-cell response. Thus, upon internalization of an EBVLP ofthe present invention, one would have expected only a CD4+ T-cellresponse but not a CD8+ T-cell response.

The term “subject” as used herein relates to an animal, preferably amammal and more preferably a human.

In a further embodiment of the invention the at least one nucleic acidencoding said Herpes viral proteins is genetically modified such that itis not packaged in the HVLPs. Usually, Herpes viruses comprisecis-acting element and proteins which are required for packaging of thevirus genome in the virus particle. Exemplarily, Herpes viruses comprisesequences at both ends of the viral DNA in its linear confirmation whichare involved in packaging, such as the “terminal repeats” (TR) ofEpstein-Barr virus and Kaposi's sarcoma-associated virus or the “asequence” of Human cytomegalovirus and Herpes simplex virus 1. By way ofexample, proteins involved in packaging of the virus genome are BFLF1,BBRF1, BGRF1, BDRF1, BALF3, BFRF1A, and BFRF1 in case of Epstein-Barrvirus, UL6, UL15, UL17, UL25, and UL28 in case of Herpes simplex virus1, UL6, UL15, UL17, UL25, UL28, UL32 and UL33 UL51 in case of Herpessimplex virus 2, UL56 and UL89 in case of Human cytomegalovirus, ORF54in case of varicella-zoster virus and ORF7, ORF29 and ORF43 in case ofKaposi's sarcoma-associated virus. Accordingly, functional inactivationof one or more of the cis-acting elements and/or proteins required forpackaging of the viral DNA results in an impaired packaging of thenucleic acid molecule and thus results in the production of HVLPs uponinduction of the lytic phase as described herein.

The term “packaging” is well-known in the art with regard to virusassembly and relates to the process of introducing the linear Herpesviral DNA into the Herpes virus particle during virus particle assemblyand specifically relates herein to the process of introducing the atleast one nucleic acid molecule into the virus particle during virusparticle assembly.

Thus, in a preferred embodiment of the invention the at least onenucleic acid encoding said Herpes viral proteins lacks a functionalcis-acting element required for packaging. The term “cis-acting elementrequired for packaging” as used herein relates to Herpes viral DNApackaging-signal sequences, which are required for packaging of theviral DNA in the virus particle. Consequently, in absence of thecis-acting element the at least one nucleic acid molecule is notpackaged upon virus synthesis during the lytic phase of the virus into awild type virus particle or a HVLP of the invention.

In a further preferred embodiment of the invention the at least onenucleic acid encoding said Herpes viral proteins comprises at least onegene encoding a Herpes viral protein required for packaging, which isgenetically modified such that said Herpes viral protein is notexpressed or non-functional, i.e. the protein required for packagingloses its packaging capacity. Thus, the gene encoding the Herpes viralprotein required for packaging may be genetically modified such that thepackaging capacity of the protein is functionally disabled while theimmunogenicity is preferably maintained. While it may be sufficient tomodify one cis-acting element or protein required for packaging suchthat it is functionally disabled, one can alternatively disable thepackaging capacity of a combination of proteins and cis-acting elementsto exclude the possibility of viral DNA packaging.

The term “genetically modified” as used herein with respect to nucleicacid sequences encoding Herpes viral proteins generally relates to anygenetic modification that renders a virus encoded protein non-functionalor prevents expression of such a protein. By way of example, such agenetic modification may be deletion of a nucleic acid sequence encodingthe functional domain or parts thereof or the entire protein. A nucleicacid sequences encoding Herpes viral proteins may further be geneticallymodified by insertion, deletion or substitution of one or morenucleotides encoding for one or more amino acids of the protein,preferably encoding the functional domain of the protein. Such amodification introduces point mutations in the coding sequencegenerating e.g. a stop codon or a shift of the open reading frame andthus results in a truncated protein. Such a modification may furtherresult in a protein with no or reduced biological function. Furthermore,expression of the gene encoding the Herpes viral protein may also beinhibited by other genetic modifications, e.g. deletion or substitutionof nucleotides of the start codon (i.e. the start codon is no longerpresent) of the open reading frame of the gene or functionallyinactivating the promoter sequence or other regulatory nucleic acidsequences required for gene expression and other methods well-known inthe art.

Accordingly, in a further embodiment the HVLP of the present inventionis substantially free of a Herpes virus genome and/or the at least onenucleic acid molecule. The term “substantially free of a Herpes virusgenome and/or the at least one nucleic acid molecule” as used hereinrelates to HVLPs that comprise less than 1000 Herpes virus genomes ornucleic acid molecules per 1 ml supernatant, less than 100 Herpes virusgenomes or nucleic acid molecules per 1 ml supernatant, less than 10Herpes virus genomes or nucleic acid molecules per 1 ml supernatant,less than 1 Herpes virus genomes or nucleic acid molecules per 1 mlsupernatant, less than 0.1 Herpes virus genomes or nucleic acidmolecules per 1 ml supernatant, less than 0.01 Herpes virus genomes ornucleic acid molecules per 1 ml supernatant, as can be easily determinedby a person skilled in the art using quantitative PCR. Preferably thedetection via quantitative PCR comprises an incubation step with DNAsein order to remove free DNA or membrane associated DNA. Accordingly, theHVLPs are preferably substantially free of DNA sequences that areidentical to Herpes virus DNA sequences, wherein said sequencespreferably relate to Herpes virus gene sequences. Furthermore, the HVLPsare preferably substantially free of nucleic acid sequences that shareat least a (for each value) 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% andat least 75% sequence identity to a wild type Herpes virus nucleic acidsequence. By way of example, HEK293 cells transfected with a B95.8 EBVgenome that lacks the TR element and the BZLF1 coding region aretransfected with an expression vector encoding BZLF1 in order to inducethe lytic phase. Three days later the supernatant can be harvested andEBVLPs can be precipitated by ultracentrifugation. The number of EBVgenomes can be quantified via quantitative RT-PCR using primers specificfor an EBV gene comprised by said B95.8 genome and by the reference EBVgenome. As a reference for quantification one can use a serial dilutionof Namalwa DNA, a human Burkitt's lymphoma cell line that contains twoEBV genome copies per cell. The quantification of EBV genome copies inEBVLPs is further described in WO2013/098364, whereas sensitivity of thedescribed quantification can be increased by using more than one mlsupernatant. In a preferred embodiment the HVLPs of the presentinvention comprise no detectable viral genomes, wherein the method fordetection is quantitative PCR.

However, as described herein at least one gene of the Herpes virusencoding for a protein that is required for inducing virus synthesis maybe genetically modified such that said Herpes viral protein (e.g. BZLF1in case of EBV) is not expressed or non-functional. In this case said atleast one gene has to be provided to the host cell in order to inducevirus synthesis for production of the HVLPs of the present invention.The at least one gene may be provided by transfecting an expressionvector comprising the at least one gene into the host cell. Thus,without being bound by theory, it may be the case that the HVLPs of thepresent invention comprise the expression vector encoding for said atleast one gene. However, it is to be understood, that the terms“substantially free of a Herpes virus genome” and “substantially free ofthe at least one nucleic acid molecule” do not relate to an expressionvector encoding the at least one gene required for inducing virussynthesis.

In another embodiment or the invention the at least one nucleic acidmolecule encoding said Herpes viral proteins comprises at least one geneencoding a Herpes viral protein required for cellular transformation,which is genetically modified such that said Herpes viral protein is notexpressed or non-functional.

Some Herpes viruses, such as Epstein-Barr virus and Kaposi'ssarcoma-associated herpesvirus are known to cause cellulartransformation and thus induce neoplastic diseases. In order to increasethe safety of a composition comprising a HVLP of the invention uponadministration to a subject, at least one gene encoding a herpesviralprotein required for cellular transformation may be genetically modifiedsuch that said Herpes viral protein is not expressed or non-functional.Thus, the gene encoding the herpesviral protein required for cellulartransformation may be genetically modified such that the transformationcapacity of the protein is functionally disabled while theimmunogenicity is preferably maintained. While it may be sufficient tomodify one protein required for cellular transformation it may bepreferable to modify more than one protein, such as 2, 3, 4, 5, 6, 7, 8,9, 10, or more proteins required for cellular transformation in order toexclude the possibility of cellular transformation. Herpes virus genes,which are required for cellular transformation are well-known in theart. By way of example, such genes are EBNA1, EBNA-LP, EBNA2, LMP1,LMP2, EBNA3A, and EBNA3C in case of Epstein-Barr virus and LANA, K13,Kaposin A, Kaposin B, Kaposin C, K1, vIL-6, vIRF-1, and vGPCR in case ofKaposi's sarcoma-associated herpesvirus.

In a further embodiment of the invention the at least one nucleic acidmolecule encoding said Herpes viral proteins comprises at least one geneencoding a Herpes viral protein required for inducing virus synthesis,which is genetically modified such that said Herpes viral protein is notexpressed or non-functional.

The life cycle of a Herpes virus comprises a latent phase in which onlya reduce set of viral genes is expressed and no progeny virus isproduced, and a lytic phase in which viral synthesis occurs and progenyvirus is released from the host cell. During lytic replication differentclasses of lytic genes are expressed and the viral genome is amplifiedto form so-called concatamers, which are eventually cleaved inunit-length linear viral genomes that are packaged in pre-formedprocapsids. Capsids containing viral DNA will undergo furtherconformational and structural changes and egress from the infected cellas enveloped viral particles. Thus, the lytic phase of the virus lifecycle is the process that leads to intracellular assembly of viralparticles. However, in case the at least one nucleic acid moleculeencoding the proteinaceous part of the HVLP lacks one or more cis-actingelements and/or proteins required of packaging, as described herein, noviral DNA is packaged upon assembly of the viral particle and thus HVLPsare produced upon induction of the lytic phase.

The lytic phase of a Herpes virus is induced and maintained uponexpression of certain Herpes viral proteins, e.g. BZFL1, BRLF1 and BMLF1in case of Epstein-Barr virus, RTA in case of Kaposi'ssarcoma-associated virus, VP16 in case of Herpes simplex virus.

As a further safety measure it may be desirable to genetically modifyone or more genes encoding a protein required for induction of the lyticphase and thus prevent viral replication from possible residual viralgenomes. Thus, the gene encoding the herpesviral protein required forinducing virus synthesis may be genetically modified such that the lyticinduction capacity of the protein is functionally disabled while theimmunogenicity is preferably maintained.

In case one or more genes required for virus synthesis are functionallyinactivated or deleted, said one or more genes have to be provided tothe host cell comprising the at least one nucleic acid molecule in orderto induce the lytic phase of the virus and thus virus synthesis and thusconfer production of the HVLPs of the invention. Said one or more genesmay be provided to the host cell by transfecting an expression vectorcomprising said one or more genes, wherein the expression vector ispreferably a stable expression vector and wherein the expression of theone or more genes required for induction of the lytic phase ispreferably inducibly regulated.

The term “inducibly regulated” or “induced expression” as used hereinrelates to any method allowing to induce the expression of a gene atwill, e.g. by using tetracycline inducible promoters, Dox-induciblepromoters, ecdysone inducible promoters or heavy metal induciblepromoters. Further suitable promoters are well-known to the personskilled in the art. Alternatively, expression can also be regulated whenby fusing the protein coding sequence to the estrogen receptor codingsequence and thus allow activation upon the addition of estrogen.

In a further embodiment of the invention the at least one nucleic acidmolecule encoding the Herpes viral proteins comprises a Herpes virusgenome, wherein said Herpes virus is selected from the group consistingof Herpes-simplex virus 1, Herpes-simplex virus 2, Varicella-zostervirus, Epstein-Barr virus, Human cytomegalovirus, Kaposi'ssarcoma-associated herpesvirus, Human herpesvirus 6, Human herpesvirus7, Bovine herpesvirus 1, Bovine herpesvirus 2, Bovine herpesvirus 3,Bovine herpesvirus 4, Bovine herpesvirus 5, and Murine gammaherpesvirus68.

Accordingly, the at least one nucleic acid molecule may comprise orconsist of a Herpes virus genome. However, the Herpes virus genome maybe genetically modified compared to a wild type Herpes virus genome asdescribed herein.

In a further embodiment of the invention the HVLP is an Epstein-Barr VLP(EBVLP), comprising Epstein-Barr virus (EBV) proteins and EBV miRNAs.

By way of example, EBV polypeptides comprised in the particle belong tothe groups of EBV structural polypeptides and EBV lytic polypeptides. Aswill be understood by the skilled person, a particular polypeptide ofEBV may belong to more than one of the above mentioned groups ofpolypeptides. In other words, an EBV polypeptide may represent astructural polypeptide as well as a lytic polypeptide. In accordancewith the invention, a structural polypeptide of EBV relates topolypeptides involved in the structural setup of the EBV. Saidpolypeptides are preferably selected from the group consisting ofmembrane polypeptides, tegument polypeptides and capsid polypeptides.EBV membrane polypeptides comprise the polypeptides selected from thegroup consisting of BALF4, BLLF1 (also termed gp350), BDLF2, BDLF3,BKRF2, BLRF1, BNLF1 (also termed LMP-1), TP (also termed LMP-2a), BXLF2,BZLF2, and any combination thereof. EBV tegument polypeptides comprisethe polypeptides selected from the group consisting of BBRF2, BGLF2,BMLF1, BNRF1, BOLF1, BPLF1, BTRF1, BVRF1, and any combination thereof.EBV capsid polypeptides comprise the polypeptides selected from thegroup consisting of BBRF1, BcLF1, BDLF1, BFRF3, and any combinationthereof. A lytic polypeptide of EBV relates to EBV polypeptides that areinvolved in the induction and maintenance of the EBV lytic phase and/orare expressed as a consequence of the induction of the lytic phase. Saidlytic polypeptides are preferably selected from the group comprising theimmediate early genes, the early genes and the late lytic genes (Kieffand Rickinson, 2007). The lytic phase is initiated by the expression ofBZLF1 and BRLF1, both immediate early proteins, followed by theexpression of the early and late proteins. Following induction, cellsthat have become permissive for virus replication undergo cytopathicchanges characteristic of herpesviruses (Kieff and Rickinson, 2007).

The invention also relates to an EBVLP comprising EBV proteins which areencoded by at least one nucleic acid molecule which still comprisesmiRNA coding loci encoding EBV miRNAs, wherein at least one of saidmiRNA coding loci is genetically modified, wherein said at least onemiRNA coding loci is selected from the group consisting of miR-BHRF1-1,miR-BHRF1-2, miR-BHRF1-3, miR-BART1, miR-BART2, miR-BART3, miR-BART4,miR-BART5, miR-BART15. Any other miRNA coding loci of EBV can be presentin an unmodified form (i.e. identical to reference strain AJ507799 incase of EBV) or can be deleted.

In case of an EBVLP the at least one genetically modified miRNA codingloci encoding EBV miRNAs, which is comprised by the at least one nucleicacid molecule, is selected from the group consisting of miR-BHRF1-1,miR-BHRF1-2, miR-BHRF1-3, miR-BART1, miR-BART2, miR-BART3, miR-BART4,miR-BART5, miR-BART15. Accordingly, at least one, at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8 of saidmiRNA coding loci can be genetically modified in any possiblecombination. In a preferred embodiment of the invention all of said EBVmiRNA coding loci are genetically modified as described herein. Thus,the present invention does not relate to EBV strain B95.8 (GenbankAccession number V01555). However, the present invention relates to anyEBV strain B95.8 which comprises a genetic modification of at least onemiRNA coding loci as described herein. In a further embodiment of theinvention in case of an EBVLP the at least one genetically modifiedmiRNA coding loci encoding EBV miRNAs, which is comprised by the atleast one nucleic acid molecule, is selected from the group consistingof miR-BHRF1-1, miR-BHRF1-2, miR-BHRF1-3, miR-BART1, miR-BART2,miR-BART3, miR-BART4, miR-BART15. In an even further preferredembodiment all EBV miRNA coding loci, as described herein, aregenetically modified.

As a feature that increases safety upon administration of an EBVLP ofthe invention, e.g. upon vaccination, at least one gene, comprised bythe at least one nucleic acid molecule, encoding an EBV protein requiredfor B-cell transformation, selected from the group consisting of EBNA1,EBNA-LP, EBNA2, LMP1, LMP2, BHRF1, BALF1, EBNA3A, and EBNA3C, isgenetically modified such that the EBV protein is not expressed ornon-functional.

The term “required for B-cell transformation” means in accordance withthe invention that the said one or more EBV polypeptides are essentialin transforming B-cells upon infection with a wild type EBV. In otherwords, in the absence of said one or more essential EBV polypeptides aB-cell is not transformed upon infection. Accordingly, the EBVLP uponfusion with the B-cell is incapable of transforming the B-cell. While itmay suffice to disable the B-cell transformation capacity of oneessential EBV polypeptide, in order to exclude the possibility of B-celltransformation, one can alternatively disable the B-cell transformationcapacity of an essential combination of EBV polypeptides to achieve thesame result achieved when only one essential polypeptide is disabled,i.e. achieve the exclusion of the possibility of B-cell transformation.Preferably, the B-cell transformation capacity of more than the oneessential EBV polypeptide or the essential combination of EBVpolypeptides is disabled. A corresponding EBV polypeptide that isessential in B-cell transformation is EBNA2 and a combination of EBVpolypeptides essential in B-cell transformation is the combination ofBHRF1 and BALF1. Disabling the B-cell transformation capacity of EBNA2or of BHRF1 and BALF1 is sufficient to exclude the possibility of B-celltransformation. Further EBV polypeptides and combinations of EBVpolypeptides that are required for B-cell transformation are LMP1,EBNA3A and EBNA3C, EBNA1 and EBNA3A, or EBNA-LP and EBNA3C. In oneembodiment the EBV genes EBNA2, LMP1, EBNA1, EBNA3A, and EBNA3C aregenetically modified such that the EBV proteins are not expressed ornon-functional. In a further embodiment all of said genes aregenetically modified such that the EBV proteins are not expressed ornon-functional.

In a further embodiment of the invention at least one gene, comprised bythe at least one nucleic acid molecule, encoding an EBV protein requiredfor inducing virus synthesis, selected from the group consisting ofBZFL1, BRLF1 and BMLF1, is genetically modified such that the EBVprotein is not expressed or non-functional, wherein said gene ispreferably BZLF1.

As a further safety measure it may be desirable to genetically modifyone or more genes encoding a protein required for induction of the lyticphase (used interchangeably herein with the terms “replicative phase”)and thus prevent virus synthesis from possible residual viral genomes.By way of example, the EBV immediate early polypeptide BZLF1 mediatesthe disruption of latent EBV infection and is generally considered thekey regulator in the induction of the lytic phase of EBV. The persistentinfection with EBV is characterized in that there is an alternation oflytic and latent phase, wherein the induction of the lytic phase is dueto the expression of BZLF1. Accordingly, upon deletion or functionallyinactivating BZLF1 induction of the lytic phase of EBV is prevented.Consequently, said one or more genes which have been deleted orfunctionally inactivated, such as BZLF1, have to be provided to the hostcell comprising the at least one nucleic acid molecule in order toinduce the lytic phase of EBV and thus virus synthesis and thus conferproduction of the EBVLPs of the invention. Said one or more genes may beprovided to the host cell by transfecting an expression vectorcomprising said one or more genes, wherein the expression vector ispreferably a stable expression vector and wherein the expression of theone or more genes required for induction of the lytic phase ispreferably inducibly regulated.

The at least one nucleic acid encoding the EBV proteins may be modifiedby deleting or functionally inactivating a cis-acting element such thatthe at least one nucleic acid is not packaged in the EBVLPs. In apreferred embodiment of the invention the at least one nucleic acidmolecule encoding said EBV proteins lacks the packaging element TR. Thepackaging of EBV genomic DNA initiates at the terminal repeats (TR) thatare directly repeated at both ends of the viral genome in its linearstate. Said terminal repeats are recognized by an enzyme termed“terminase”. Thus, the at least one nucleic acid molecule or an EBVgenome is not packaged into the procapsid of EBV if it lacks thepackaging element TR, resulting in the production of the EBVLP of thepresent invention. In case the at least one nucleic acid molecule or theEBV genome used in the production of the EBVLP lacks the packagingelement TR, the EBV gene BALF4 may not be co-expressed in the host cellused in the production of the EBVLP.

In a further embodiment of the invention the at least one nucleic acidmolecule encoding the EBV proteins comprises at least one gene encodingan EBV protein required for packaging of EBV DNA, selected from thegroup consisting of BFLF1, BBRF1, BGRF1, BDRF1, BALF3, BFRF1A, andBFRF1, which is genetically modified such that said EBV protein is notexpressed or non-functional, wherein BFRF1A is preferred. The proteinsof said genes are required for packaging of viral DNA (i.e. an EBVgenome or the at least one nucleic acid molecule) into procapsids ofEBV. Accordingly, the at least one nucleic acid molecule or an EBVgenome is not packaged in the into the procapsid of EBV if one or moreof said genes are genetically modified such that said EBV protein is notexpressed or non-functional and thus results in the production of theEBVLP of the present invention.

In a further embodiment of the invention the at least one nucleic acidmolecule encoding said EBV proteins comprises an EBV genome. In thiscase, the at least one nucleic acid molecule encoding the EBVLP maycomprise or consist of an EBV genome, which may be genetically modifiedcompared to a wild type EBV genome as described herein and wherein theat least one nucleic acid molecule encoding the EBVLP still comprisesEBV encoded miRNAs.

Exemplarily, the publications Delecluse et al. (PNAS, vol. 96, pp.5188-5193, 1999) and Ruiss et al. (Journal of Virology, pp. 13105-13113,2011) of the present inventors and the documents WO2012/025603 andWO2013/098364 disclose nucleic acid molecules that confer the productionof an EBVLP upon expression in a suitable cell line, e.g. HEK293 cells.Said nucleic acid molecules have been modified in accordance with thepresent invention but still comprise miRNA encoding loci. Accordingly, anucleic acid molecule of the present invention can be obtained by usingone of said nucleic acid molecules disclosed in the publications anddocuments cited above and deleting at least one miRNA encoding loci asdescribed herein, wherein the obtained nucleic acid molecule can befurther modified as described herein.

The present invention further pertains to a nucleic acid moleculeencoding the Herpes viral proteins of the HVLP or the EBV proteins ofthe EBVLP. The present invention also pertains to a vector comprisingthe nucleic acid molecule encoding the Herpes viral proteins of the HVLPor the EBV proteins of the EBVLP.

The term “vector” as used herein with respect to the at least onenucleic acid molecule encoding the Herpes virus proteins or the EBVproteins refers to a nucleic acid sequence into which one or moreexpression cassettes comprising a gene encoding the protein of interestmay be inserted or cloned. Furthermore, the vector preferably encodes anantibiotic resistance gene conferring selection of the host cell and/ora phenotypical marker, e.g. a fluorescent protein, such as GFP, RFP,YFP, BFP or others. Preferably, the vector is a plasmid or viral vector.The vector can contain elements for propagation in bacteria (e.g. E.coli), yeast (e.g. S. cerevisiae), insect cells and/or mammalian cells.Preferably, said vector comprises a bacterial mini-F-factor plasmidelement, allowing propagation in E. coli.

In a further embodiment the present invention provides a composition ofmatter comprising at least two nucleic acid molecules encoding theHerpes viral proteins of the HVLP or the EBV proteins of the EBVLP. Inan even further embodiment of the present invention the at least twonucleic acid molecules are comprised by at least two vectors (i.e. twonucleic acid molecules are comprised by two vectors, three nucleic acidmolecules are comprised by three vectors, four nucleic acid moleculesare comprised by four vectors, etc.).

The present invention further provides a host cell transfected with thenucleic acid molecule encoding the Herpes viral proteins of the HVLP orthe EBV proteins of the EBVLP or the vector comprising said nucleic acidmolecule or the composition of matter comprising at least two nucleicacid molecules encoding the Herpes viral proteins of the HVLP or the EBVproteins of the EBVLP, wherein the at least two nucleic acid moleculesare preferably comprised by at least two vectors.

The term “host cell” as used herein relates to a cell, which allowslytic replication of the Herpes virus or EBV resulting in the formationof HVLPs or EBVLPs. Such a host cell is preferably a mammalian cell,more preferably a primate cell, even more preferably a human cell andmost preferably a HEK293 cell. In case the Herpes virus or EBV lack oneor more functional proteins required for inducing virus synthesis (i.e.the lytic phase), it is envisioned that the host cell may provide theone or more proteins (e.g. BZLF1 in case of EBV). The host cell mayprovide the one or more proteins via a transfected vector, a stablytransfected vector or by chromosomal integration of the nucleic acidsequence encoding the one or more proteins, wherein expression of saidone or more proteins is preferably inducibly regulated.

The term “transfection” as used herein relates to the process ofintroducing nucleic acids into cells. Transfection can be achieved by avariety of methods such as, e.g. chemical-based methods like calciumphosphate-mediated transfection or liposome-mediated transfection(lipofection). Also non-chemical methods like electroporation orsonoporation or particle-based methods such as gene-gun-mediatedtransfection or magnetofection as well as viral-mediated methods areknown in the art.

The present invention further pertains to a method for generating a HVLPor an EBVLP, the method comprising:

-   -   (i) culturing the host cell under conditions that allow        expression of the Herpes viral proteins or the EBV proteins; and    -   (ii) obtaining said HVLP or EBVLP.

The present invention further relates to a HVLP or an EBVLP obtainableby the method for generating a HVLP or an EBVLP of the presentinvention. The present invention also relates to a vaccine comprising aHVLP or an EBVLP obtainable by the method for generating a HVLP or anEBVLP of the present invention.

The method of the present invention is preferably an in vitro method.Exemplarily, the generation of EBVLPs is disclosed in the publicationsDelecluse et al. (PNAS, vol. 96, pp. 5188-5193, 1999) and Ruiss et al.(Journal of Virology, pp. 13105-13113, 2011) of the present inventorsand in the documents WO2012/025603 and WO2013/098364.

The term “culturing” as used herein relates to growing cells outside theorganism in cell culture medium and is known by the person skilled inthe art. Suitable cell culture media confer survival and replication bythe cells and are commercially available. They may comprise nutrients,salts, growth factors, antibiotics, serum (e.g. fetal calf serum) andpH-indicators (e.g. phenol red).

The term “obtaining” as used herein relates to isolating and/orpurifying the HVLPs or EBVLPs, preferably from the cell culturesupernatant. Such isolation and/or purification steps are known to theperson skilled in the art and encompass for example methods such asdensity gradient centrifugation, size-exclusion chromatography, affinitychromatography, precipitation and in case of EBV by binding EBVLP tomagnetic beads via anti-gp350 antibodies.

The method of the present invention may further comprise after step (i)and prior to step (ii) a further step (i′), comprising inducing thereplicative phase of the Herpes virus or Epstein-Barr virus, whereinsaid replicative phase is induced by expressing at least one gene,encoding a Herpes viral protein or an EBV protein that is required forinducing Herpes virus synthesis or EBV synthesis, wherein said Herpesviral protein or EBV protein has been genetically modified in the atleast one nucleic acid molecule encoding the Herpes viral proteins ofthe HVLP or the EBV proteins of the EBVLP, such that it is not expressedor non-functional.

In a further embodiment of the method of the present invention the atleast one gene encoding a Herpes viral protein or an EBV protein that isrequired for inducing Herpes virus synthesis or EBV synthesis isexpressed from a stably transfected vector in said host cell and/orwherein said gene is inducibly regulated. In a further preferredembodiment of the method of the present invention the at least one geneencoding an EBV protein that is required for inducing EBV synthesis isselected from the group consisting of BZLF1, BRLF1 and BMLF1, whereinBZLF1 is preferred.

In a further embodiment the present invention relates to a compositioncomprising at least 99.99%, 99.9%, 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%,78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, 60%, 55%, 50%, 45%,40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or preferably 95% of the HVLP orthe EBVLP as defined herein. Accordingly, such a composition may furthercomprise, irrespective of the comprised HVLPs or the EBVLPs as describedherein, HVLPs or EBVLPs that are not in line with the descriptionherein, e.g. such a composition may comprise defect HVLPs or EBVLPs thatare not morphologically similar to a virus particle, as can bedetermined using electron microscopy.

In another embodiment the present invention pertains to a vaccinecomposition comprising the HVLP or the EBVLP as described herein or thecomposition comprising the HVLPs or the EBVLPs as described herein.

The terms “vaccine” and “vaccine composition” are used interchangeablyherein and relate to a composition comprising HVLPs or EBVLPs of thepresent invention which -when administered to a subject-elicits animmune response against the Herpes virus or EBV. Thus, administeringsaid vaccine composition to a subject stimulates the immune system andestablishes or improves immunity to a new and/or persisting infectionwith the Herpes virus or EBV. Preferably, the vaccine according to thepresent invention allows for establishing or improving immunity to a newand/or persisting infection with EBV. Preferably, the immunizationcauses activation and expansion of T-cells and/or B-cells specificallyrecognizing EBV antigens, e.g. EBV structural antigens. Even morepreferably, the immunization causes activation and expansion of CD8+T-cells. It is also preferred that, immunization causes the productionof antibodies preventing infection of body cells by EBV.

In a further embodiment the vaccine composition of the present inventionfurther comprises an excipient.

The terms “carrier” and “excipient” are used interchangeably herein.Pharmaceutically acceptable carriers include, but are not limited todiluents (fillers, bulking agents, e.g. lactose, microcrystallinecellulose), disintegrants (e.g. sodium starch glycolate, croscarmellosesodium), binders (e.g. PVP, HPMC), lubricants (e.g. magnesium stearate),glidants (e.g. colloidal SiO2), solvents/co-solvents (e.g. aqueousvehicle, Propylene glycol, glycerol), buffering agents (e.g. citrate,gluconates, lactates), preservatives (e.g. Na benzoate, parabens (Me, Prand Bu), BKC), anti-oxidants (e.g. BHT, BHA, Ascorbic acid), wettingagents (e.g. polysorbates, sorbitan esters), anti-foaming agents (e.g.Simethicone), thickening agents (e.g. methylcellulose orhydroxyethylcellulose), sweetening agents (e.g. sorbitol, saccharin,aspartame, acesulfame), flavouring agents (e.g. peppermint, lemon oils,butterscotch, etc), humectants (e.g. propylene, glycol, glycerol,sorbitol). Further pharmaceutically acceptable carriers are(biodegradable) liposomes; microspheres made of the biodegradablepolymer poly(D,L)-lactic-coglycolic acid (PLGA), albumin microspheres;synthetic polymers (soluble); nanofibers, protein-DNA complexes; proteinconjugates; erythrocytes; or virosomes. Various carrier based dosageforms comprise solid lipid nanoparticles (SLNs), polymericnanoparticles, ceramic nanoparticles, hydrogel nanoparticles,copolymerized peptide nanoparticles, nanocrystals and nanosuspensions,nanocrystals, nanotubes and nanowires, functionalized nanocarriers,nanospheres, nanocapsules, liposomes, lipid emulsions, lipidmicrotubules/microcylinders, lipid microbubbles, lipospheres,lipopolyplexes, inverse lipid micelles, dendrimers, ethosomes,multicomposite ultrathin capsules, aquasomes, pharmacosomes,colloidosomes, niosomes, discomes, proniosomes, microspheres,microemulsions and polymeric micelles. Other suitable pharmaceuticallyacceptable excipients are inter alia described in Remington'sPharmaceutical Sciences, 15th Ed., Mack Publishing Co., New Jersey(1991) and Bauer et al., Pharmazeutische Technologie, 5th Ed.,Govi-Verlag Frankfurt (1997). The person skilled in the art will readilybe able to choose suitable pharmaceutically acceptable carriers,depending, e.g., on the formulation and administration route of thepharmaceutical composition.

In a further embodiment the vaccine composition comprises one or moreviral or non-viral polypeptides, one or more viral or non-viral nucleicacid sequences and/or vaccine adjuvants, wherein said one or more viralpolypeptides or said one or more viral nucleic acid sequences are notfrom the same virus as the HVLP or EBVLP in said vaccine composition.

The term “adjuvant” as used herein refers to a substance that enhances,augments or potentiates the host's immune response (antibody and/orcell-mediated) to an antigen or fragment thereof. Exemplary adjuvantsfor use in accordance with the present invention include inorganiccompounds such as alum, aluminum hydroxide, aluminum phosphate, calciumphosphate hydroxide, the TLR9 agonist CpG oligodeoxynucleotide, the TLR4agonist monophosphoryl lipid (MPL), the TLR4 agonist glucopyranosyllipid (GLA), the water in oil emulsions Montanide ISA 51 and 720,mineral oils, such as paraffin oil, virosomes, bacterial products, suchas killed bacteria Bordetella pertussis, Mycobacterium bovis, toxoids,nonbacterial organics, such as squalene, thimerosal, detergents (QuilA), cytokines, such as IL-1, IL-2, IL-10 and IL-12, and complexcompositions such as Freund's complete adjuvant, and Freund's incompleteadjuvant. Generally, the adjuvant used in accordance with the presentinvention preferably potentiates the immune response to the multimericcomplex of the invention and/or modulates it towards the desired immuneresponses.

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe multimeric complex according to the present invention.

In a further embodiment the present invention relates to the use of theHVLP or the EBVLP as described herein or the composition comprising theHVLP or the EBVLP as described herein, or the vaccine compositioncomprising the HVLP or the EBVLP as described herein in the vaccinationor treatment of a subject.

The amount necessary and the treatment regimen for an effectiveimmunization may vary and depend on such factors as the individual'ssize, body surface area, age, sex, time and route of administration,general health, and other drugs being administered concurrently. Saideffective amount is expected to be in broad range and can for any givensituation be readily determined by routine experimentation and is withinthe skills and judgement of the ordinary clinician or physician. Themode of administration can be any mode of administration that results inthe immunization of the individual exposed to the vaccine forimmunization and includes parenteral administration such as, e.g.,intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous,intraarticular injection or infusion and inhalation, as well as enteraladministration. Preferably, the vaccine is administered at least 2 timesin order to maximize the effect of the immunization.

The term “vaccination” as used herein relates to the administration ofantigenic material to a subject in order to stimulate the immune systemof the subject in order to prophylactically or therapeutically immunizethe subject against a Herpes virus or EBV infection or diseasesassociated with the viruses. According to the invention, prophylacticimmunization refers to the first exposure of an individual's immunesystem, i.e. a naive immune system, to Herpes virus or EBV antigens.Said first exposure results in the clearance of said antigens from thebody of the exposed individual and in the development of Herpes virus-or EBV-antigen specific CD4+ and CD8+ T-cells and antibody-producingmemory B-cells. Upon a second exposure the immune system is able toprevent Herpes virus or EBV infection and/or clear said infection moreeffectively thereby preventing or mitigating the development of Herpesvirus- or EBV-associated diseases. Specifically, the effects of saidprophylactic immunization manifest itself in at least one of thefollowing: preventing infection of the immunized individual with theHerpes virus or EBV, modifying or limiting the infection, aiding,improving, enhancing or stimulating the recovery of said individual frominfection and generating immunological memory that will prevent or limita subsequent Herpes virus or EBV infection. The presence of any of saideffects can be tested for and detected by routine methods known to theperson skilled in the art. Preferably, the patient is challenged withone or more Herpes virus or EBV antigens which have been part of thevaccine used and antibody titers and the number of T-cells against saidone or more antigens are determined. Also, the induction of neutralizingantibodies that inhibit infection of human B-cells in vitro can bedetermined. While equally provoking an immune response against Herpesvirus or EBV antigens, therapeutic immunization in accordance with thepresent invention is performed on individuals that have been exposed tothe Herpes virus or EBV prior to said immunization, i.e. they arealready infected with the Herpes virus or EBV. In this case,immunization leads to the reactivation of resting T effector cells,which are confronted with the cognate antigens in a form that theseantigens are presented by professional antigen-presenting cells inassociation with MHC class I and/or MHC class II molecules. Therapeuticimmunization against EBV may prove particularly relevant in cases wherethe reactivation of the virus is undesirable such as, e.g. in transplantrecipients or otherwise immunocompromised patients (HIV-positiveindividuals, cancer patients, patients with severe inflammatory orautoimmune diseases), or in cases where EBV-reactivation can lead to orhas led to the development of a disease like posttransplantlymphoproliferative disorders (PTLD) and Non-Hodgkin lymphoma, chronicactive EBV infection (CAEBV), oral hairy leukoplakia or in cases wherethe B-cell transforming capacity of EBV has led to the development of adisease such as, e.g. cancer.

In a further embodiment the present invention relates to the use of thenucleic acid molecule encoding the Herpes viral proteins of the HVLP orthe EBV proteins of the EBVLP, the vector comprising said nucleic acidmolecule, the composition of matter comprising at least two nucleic acidmolecules encoding the Herpes viral proteins of the HVLP or the EBVproteins of the EBVLP, wherein the at least two nucleic acid moleculesare preferably comprised by at least two vectors, the host celltransfected with said nucleic acid molecule, said vector or saidcomposition in the production of a HVLP or an EBVLP.

The present invention further pertains to a kit comprising the HVLP asdescribed herein, the EBVLP as described herein, the nucleic acidmolecule encoding the Herpes viral proteins of the HVLP or the EBVproteins of the EBVLP, the vector comprising said nucleic acid molecule,the composition of matter comprising at least two nucleic acid moleculesencoding the Herpes viral proteins of the HVLP or the EBV proteins ofthe EBVLP, wherein the at least two nucleic acid molecules arepreferably comprised by at least two vectors, the host cell transfectedwith said nucleic acid molecule, said vector or said composition, thecomposition comprising at least 95% of the HVLP or the EBVLP asdescribed herein and/or the vaccine composition as described herein.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or sometimes when used herein with the term “having”. Whenused herein “consisting of” excludes any element, step, or ingredientnot specified in the claim element. When used herein, “consistingessentially of” does not exclude materials or steps that do notmaterially affect the basic and novel characteristics of the claim. Ineach instance herein any of the terms “comprising”, “consistingessentially of” and “consisting of” may be replaced with either of theother two terms.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange. It includes also the concrete number, e.g., about 20 includes 20.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present invention are generally performed according toconventional methods well-known in the art. Generally, nomenclaturesused in connection with techniques of biochemistry, enzymology,molecular and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well-known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e. g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N. Y. (2001); Ausubel et al., Current Protocols in MolecularBiology, J, Greene Publishing Associates (1992, and Supplements to2002); Handbook of Biochemistry: Section A Proteins, Vol 11976 CRCPress; Handbook of Biochemistry: Section A Proteins, Vol II 1976 CRCPress. The nomenclatures used in connection with, and the laboratoryprocedures and techniques of, molecular and cellular biology, proteinbiochemistry, enzymology and medicinal and pharmaceutical chemistrydescribed herein are those well-known and commonly used in the art.

FIGURES

FIG. 1 : EBV miRNAs affect major pathways of immunity. (A) Heatmaps ofthe most strongly regulated genes in wt/B95-8 or ΔmiR EBV-infectedB-cells of 6 donors (donor Ad1-Ad6) five days post infection.Differentially expressed gene transcripts with absolute z-scores >1.6are shown. Blue and red colors indicate down- and up-regulatedtranscripts, respectively, in wt/B95-8 compared with ΔmiR EBV-infectedcells. (B) Regulation of selected genes associated with adaptive immuneresponses or the p53 signaling pathway. Previously reported targets ofEBV miRNAs and common housekeeping genes are shown as well. Bluebackground shadings indicate genes down-regulated by viral miRNAs. (C)The fractions of EBV miRNAs among all miRNAs. Means of 6 donors isshown.

FIG. 2 : EBV miRNAs inhibit secretion of pro-inflammatory cytokines andexpression of molecules involved in antigen processing and presentation.(A) Secretion of various cytokines by B-cells infected with wt/B95-8 orΔmiR EBV. B-cells, which had been infected 4 or 11 earlier (days postinfection, dpi), were cultivated for 4 additional days to determinecytokines levels by ELISA (n=3). CpG DNA was added as indicated. (B) EBVmiRNAs regulate IL12B and TAP2. HEK293T cells were co-transfected withmiRNA expression vectors and luciferase reporter plasmids carrying awild type or mutated 3′-UTR (FIG. 7 ) as indicated (n=3). The luciferaseactivities were normalized to lysates from cells co-transfected with thewild type 3′-UTR reporter and an empty plasmid. wt: wild type 3′-UTR,mut: mutated 3′-UTR, ø: empty plasmid. P values were calculated by anunpaired two-tailed T test. An asterisk (*) indicates p<0.05 withrespect to the luciferase activity of the wild type reporterco-transfected with empty plasmid. (C) Western blot analysis of TAP1 andTAP2 in EBV-infected B-cells. Tubulin (TUBB) and β-Actin (ACTB) serve ashousekeeping controls. A positive control is IPO7. Representativeexamples (top) and protein expressions normalized to tubulin (bottom;n=3-5) are shown. (D-E) Cell surface expression of HLA molecules (D) andco-stimulatory and adhesion molecules (E) regulated by EBV miRNAs.Median fluorescence intensity (MFI) was measured after immunostainingsfor individual surface proteins and ratios (wt/B95-8 divided by ΔmiREBV-infected B-cells) are shown (n=5-10). Means±SD are shown. n.d.: notdetected; wt: wt/B95-8; *: p<0.05, **: p<0.01, ***: p<0.001.

FIG. 3 : EBV miRNAs prevent Th1 differentiation and recognition byEBV-specific CD4⁺ T-cells. (A) Schematic overview of co-cultureexperiments to assess the impact of viral miRNAs on helper T-celldifferentiation. (B) Th1 differentiation of naive CD4⁺ T-cells uponco-culture with EBV-infected B-cells. Naive CD4⁺ T-cells were cultivatedfor 7 days with autologous, newly infected B-cells and αCD3/αCD28antibodies at indicated ratios (n=5-6). Proliferating, phorbol12-myristate 13-acetate (PMA) and ionomycin re-stimulated Th1 cells werequantitated by intracellular IFN-γ staining. Left: representative flowcytometry analyses; right: summary of all experiments. (C) An anti-IL12Bantibody (5 μg/ml) suppressed Th1 cell differentiation of naive CD4⁺T-cells co-cultivated with wt/B95-8 or ΔmiR-infected B-cells at B:T-cellratio of 1:1 (n=8). An irrelevant antibody of the same isotype was usedas a control. (D) Schematic overview of co-culture experimentsinvestigating the influence of viral miRNAs on antiviral functions ofEBV-specific CD4⁺ T-cells. (E) IFN-γ release by polyclonal EBV-specificCD4⁺ T-cells co-cultured with autologous (auto), HLA-matched, ormismatched (mis.) B-cells infected with EBV (n=3; FIG. 12 ). TheB:T-cell ratio was 1:1. Matched HLA class II alleles are indicated. ø:only T-cells; n.a.: not applicable. (F) Cytotoxic activity ofEBV-specific CD4⁺ T-cells. Killing of EBV-infected B-cells was analyzedat various B:T-cell ratios by Calcein release assays. A representativeexperiment with HLA-matched EBV-infected target B-cells (left; n=3) andthe overview of all experiments with HLA-matched B-cells (right) aredescribed. Means±SD are shown. *: p<0.05, **: p<0.01, ***: p<0.001.

FIG. 4 : EBV miRNAs inhibit recognition of EBV-infected B-cells byEBV-specific CD8⁺ T-cells. (A) Schematic overview of co-cultureexperiments investigating the influence of viral miRNAs on antiviralfunctions of EBV-specific CD8⁺ T-cells. (B) IFN-γ release by polyclonalEBV-specific CD8⁺ T-cells co-cultured with autologous (auto),HLA-matched, or mismatched (mis.) B-cells infected with EBV (n=3; FIG.12 ). The B:T-cell ratio was 1:1. Matched HLA class I alleles areindicated. ø: only T-cells; n.a.: not applicable. (C) Cytotoxic activityof EBV-specific CD8⁺ T-cells. Killing of EBV-infected B-cells (wt/B95-8or ΔmiR EBV) was analyzed at various B:T-cell ratios in calcein releaseassays. A representative experiment with HLA-matched EBV-infected targetB-cells (left; n=3) and the overview of all experiments with HLA-matchedB-cells (right) are shown. (D) Reactivity of a CD8⁺ T-cell clonedirected against a LMP2 epitope IED (HLA-B*40:01-restricted). T-cellswere cultivated for 16 hours with HLA-B*40:01-positive B-cells that havebeen infected for 15 days. IFN-γ secretion levels quantified with ELISA(Left; n=3) and MFI ratios (wt/B95-8 divided by ΔmiR EBV-infectedB-cells) for HLA-B*40 (Right; n=4) are described. ø: only T-cells;peptide: T-cells loaded with the control peptide. wt: wt/B95-8. Means±SDare shown. *: p<0.05, **: p<0.01.

FIG. 5 : The regulation of functional gene groups by EBV miRNAs

KEGG pathway categories were used for categorization of gene functions.Pathways are sorted by statistical significance. The sizes of the orangedots indicate-log10 p-value scores. For each of the six donors, foldchange values of differentially expressed transcripts are plotted. As inFIG. 1 a, blue or red colors indicate down- or up-regulation by EBVmiRNAs, respectively.

FIG. 6 : Quantification of EBV miRNAs after RISC-IP

EBV's BHRF and BART miRNAs accumulate in wt/B95-8 EBV-infected B-cellsbut are barely detectable in ΔmiR EBV-infected B-cells. Means±SD isshown.

FIG. 7 : 3′-UTR reporters and their mutations

Partial sequences of 3′-UTRs of selected transcripts (IL12B targetsequences BART2; SEQ ID NOs: 2 and 4; ID 2B target sequences BART10; SEQID NOs: 7 and 9; ID 2B target sequence BART22; SEQ ID NO: 12; TAP2target sequence BHRF1-3; SEQ ID NO: 15; IL12B target sequence BART1; SEQID NO: 18; and TAP2 target sequences BART17; SEQ ID NOs: 21 and 23),which were analyzed in FIG. 2 b are shown together with correspondingmiRNAs (BART2 miRNA; SEQ ID NO: 1; BART10 miRNA; SEQ ID No: 6, BART22miRNA; SEQ ID NO: 11; BHRF1-3 miRNA; SEQ ID NO: 14; BART1 miRNA; SEQ IDNO: 17; and BART17 miRNA; SEQ ID NO. 20) and mutations (mutated IL12Btarget sequences BART2; SEQ ID NOs: 3 and 5; mutated IL12B targetsequences BART10; SEQ ID NOs: 8 and 10; mutated IL12B target sequenceBART22; SEQ ID No: 13; mutated TAP2 target sequence BHRF1-3; SEQ ID NO:16; mutated IL12B target sequence BART1; SEQ ID NO: 19; and mutated TAP2target sequences BART17; SEQ ID NOs: 22 and 24) within the 3′-UTRs inreporter vectors. Complementarities are based on in silico predictionsaccording to the RNAhybrid algorithm and depicted as Watson-Click (‘|’)or G:U (′:′). Non-matching nucleotide residues are indicated (X). Theyresult from mutated mRNA target sequences in the reporter plasmids.

FIG. 8 : Reactivity of polyclonal EBV-specific CD4⁺ T-cells

EBV-specific CD4⁺ T-cell were co-cultured for 16 hours with autologousB-cells that had been infected five days earlier. IFN-γ secretion levelswere then quantified with ELISA.Various B:T-cell ratios were used asindicated.

FIG. 9 : Reactivity of the gp350 specific CD4⁺ T-cell clone

The gp350-specific CD4⁺ T-cell clone, epitope FGQ (HLA-DRB1*1301), wasused as effector cells. Autologous B-cells from donor JM (table S2) wereused as target cells five and 15 days after infection with the two EBVstrains indicated at an B:T-cell ratio of 1:1. After 16 hours ofco-culture, IFN-γ and GM-CSF secretion levels were quantified by ELISA.Means±SD are shown.

FIG. 10 : Schematic overview of co-culture experiments investigating theinfluence of viral miRNAs on antigen presentation to a LMP2-specificCD8⁺ T-cells clone

FIG. 11 : Regulation of viral genes by EBV miRNAs

(A) Western blot analysis of LMP2A expression in B-cells infected withwt/B95-8 or with ΔmiR EBV at day 15 post infection. A representativeexample (top) and protein expression normalized to tubulin (bottom n=4)are described. Means±SD are shown. (B) Loge fold changes of two LMP2gene variants by viral miRNAs. Analysis was performed as in FIG. 1B butthe quantification of expression level was done exon-wise to analysesplicing variants correctly.

FIG. 12 : HLA alleles.

List of the donors' HLA alleles (MVZ Martinsried, Germany) identified bydeep-sequencing, whose B and T-cells have been used in co-cultureexperiments in this study. n.a.: not available.

FIG. 13 : Activation of CD4⁺ T-cells using Epstein-Barr VLPs A humanEBV-immortalized B-cell line (LCL) was incubated with similar numbers ofVLPs (1*10{circumflex over ( )}4 particles/cell) with miRNAs or lackingall miRNAs (ΔmiRNAs) for 24 hours and then co-cultivated for another 24hours with an HLA-matched CD4⁺ T-cell clone specific for the EBVtegument protein BNRF1 for another 24 h. Activation of T cells wasquantified in an IFNγ-ELISA assay. Controls are LCLs or T-cells thathave not been co-cultivated with LCLs (T-cells only).

EXAMPLES

The following Examples illustrate the invention, but are not to beconstrued as limiting the scope of the invention.

Materials and Methods

Separation of Human Primary Cells

Human primary B and T-cells were prepared from adenoidal mononuclearcells (MNC) or peripheral blood mononuclear cells (PBMC) byFicoll-Hypaque gradient centrifugation. B-cells, CD4⁺ T-cells, CD8⁺T-cells, and naive CD4⁺ T-cells were separated from adenoidal MNC orPBMC using MACS separator (Miltenyi Biotec) with CD19 MicroBeads, CD4MicroBeads, CD8 MicroBeads, and Naive CD4⁺ T-cell Isolation Kit II,respectively.

Cell Lines and Cell Culture

The EBV-positive Burkitt's lymphoma cell line Raji and HEK293-based EBVproducer cell lines (Seto et al., PLoS Pathog. 6, e1001063 (2010)),infected human primary B-cells, and isolated T-cells were maintained inRPMI 1640 medium (Life Technologies). HEK293T cells were maintained inDMEM medium. All media were supplemented with 10% FBS (LifeTechnologies), penicillin (100 U/ml; Life Technologies), andstreptomycin (100 mg/ml; Life Technologies). Cells were cultivated at37° C. in a 5% CO₂ incubator.

Preparation of Infectious EBV Stocks and Infection of Human PrimaryB-Cells

Infectious EBV stocks were prepared as described (Seto, loc. cit.).Briefly, EBV producer cell lines for ΔmiR (4027) and wt/B95-8 (2089) EBVstrains were transiently transfected with expression plasmids encodingBZLF1 and BALF4 to induce EBV's lytic phase. Supernatants were collectedthree days after transfection and debris was cleared by centrifugationat 3000 rpm for 15 minutes. Virus stocks were titered on Raji cells aspreviously reported (Seto, loc. cit.). For virus infection, primaryB-cells were cultivated with each virus stock for 18 hours. Afterreplacement with fresh medium, the infected cells were seeded at aninitial density of 5×10⁵ cells per ml.

RNA-Seq and RISC-IP

At 5 days post infection of human primary B-cells, total RNAs wereextracted with Trizol (Life Technologies) and Direct-Zol RNA MiniPrep(Zymo Research) from six different donors (Ad1 to Ad6) (FIG. 1 ) forRNA-Seq, according to the manufacturers' protocols. In parallel, RISCimmunoprecipitation (RISC-IP) was performed as described previously(Kuzembayeva, et al., PLoS ONE. 7, e47409 (2012)). Briefly, lysed cellswere incubated with anti-Ago2 antibody (11A9)-conjugated dynabeads (LifeTechnologies), washed, and co-precipitated RNA was extracted. The cDNAlibraries were prepared (vertis Biotechnologie AG, Freising, Germany).For RNA-Seq, total RNAs were depleted of rRNAs by Ribo-Zero rRNA RemovalKit (Illumina), fragmented by ultrasonication, and subjected to firststrand synthesis with a randomized primer. For RISC-IP, RNAs were poly(A)-tailed, ligated with an RNA adapter at 5′-phosphates to facilitateIllumina TruSeq sequencing, and subjected to first strand synthesis witha oligo-(dT) primer. The cDNAs were PCR-amplified and sequenced with anIllumina HiSeq2000 instrument at the University of WisconsinBiotechnology Center DNA Sequencing Facility.

Analysis of Deep Sequencing

For RNA-Seq, processing of paired-end reads (poly-A tail filtering,N-filtering, adapter removal) was done using FastQC and R2M(RawReadManipulator). Reads were mapped to the human genome (hg19 ‘core’chromosome-set) by STAR and feature counts per transcript weredetermined using featureCounts and GencodeCV19 annotations together withEBV's annotation (GenBank: AJ507799). To screen differentially regulatedgenes by viral miRNAs, it was used a simple but efficient scoringalgorithm based on donor/replicate wise fold changes ranks. For eachgene g and replicate k it is calculated the gene specific rank score:

$r_{g} = {\frac{1}{m}{\sum\limits_{k = 1}^{n}r_{gk}}}$

where n is the number of all replicates, m the number of allgenes/transcripts, r_(gk) the rank of gene g in sample k.

To select highly differentially expressed genes the rank score wastransformed into a z-score and selected all transcripts with an absolutez-score>1.6.

For RISC-IP the mapped reads were normalized using size factorsestimated with the R package DEseq2 and filtered for reads mapped toannotated 3′UTR regions using Gencode v19. To identify localquantitative differences in the read enrichments on 3′UTRs between wtEBV compared with ΔmiR EBV-infected B cells, a donor-wise relativeenrichment score was calculated. For each genomic position p, therelative expression es_(p) was calculated as:

${es}_{p} = {\frac{e_{tp}}{e_{tp} + e_{cp}} \cdot n_{pu}}$

where e_(tp) is the expression value at position p in wt EBV-infectedcells and e_(cp) the local expression value in ΔmiR EBV-infected Bcells, respectively.

The normalization factor n_(pu)=e_(tp)/max(e_(u)) was introduced tocorrect for local maxima in the UTR sequence of interest, wheremax(e_(u)) is the maximum expression value in the UTR sequence u.Finally a Gaussian filter was used to minimize local noise. To select3′-UTRs bound by viral miRNAs, the threshold was set as follows:enrichment score>0.6 for a stretch of >20 nucleotides in the 3′-UTRs intwo or more donors.

KEGG Enrichment Pathway

Enrichment of specific pathways was estimated by performing ahypergeometric distribution test via the KEGG API Web Service. Allcalculations were done using Matlab (Mathworks).

ELISA

To detect cytokine secretion from infected B-cells, 1×10⁶ cells wereseeded in 6 well plates at four or 11 days post infection, cultivatedfor four days with cyclosporine (1 μg/ml; Novartis). Supernatants wereharvested and stored at −20° C. Enzyme-linked immunosorbent assays(ELISAs) for interleukin-6 (IL-6), IL-10, IL12B (IL-12p40), IL-12,IL-23, and TNF-α were performed following the manufacturer's protocols(Mabtech). For IL-6, IL-10, and TNF-α, CpG DNA were added as previouslydescribed (Iskra, et al., J. Virol. 84, 3612-3623 (2010)) to stimulateinfected B-cells. ELISA for IFN-γ levels was performed following themanufacturer's protocol (Mabtech).

Flow Cytometry and Antibodies

After immunostainings with fluorophore-conjugated antibodies,single-cell suspensions were measured with LSRFortessa or FACSCanto (BD)flow cytometers and the FACSDiva software (BD Biosciences). Acquireddata were analyzed with FlowJo software Ver. 9.8 (FlowJo). The followingfluorophore-conjugated antibodies reactive to human antigens were used:anti-human IFN-γ APC (4S.B3, IgG1; Biolegend), anti-CD40 PE (5c3, IgG2b;BioLegend), anti-ICOS-L (B7-H2) PE (2D3, IgG2b; BioLegend), anti-PD-L1(B7-H1) APC (29E.2A3, IgG2b; BioLegend), anti-CD86 (B7-2) PE (37301,IgG1; R&D Systems), anti-CD54 (ICAM-1) APC (HCD54, IgG1; BioLegend),anti-HLA-ABC APC (W6/32, IgG2a; BioLegend), anti-CD80 PE-Cy5 (L307.4; BDPharmingen), anti-FAS (CD45) PE (Dx2, IgG1; BioLegend), anti-HLA-DRunlabeled (L234, IgG2a; BioLegend), anti-HLA-DQ unlabeled (SPV-L3,IgG2a; AbD Serotec), anti-HLA-DP unlabeled (B7/21, IgG3; Abcam),anti-mouse F(ab′)2 APC (polyclonal, IgG; eBioscience), HLA-Bw6 PE(REA143, IgG1; Miltenyi Biotec), isotype IgG1 PE (MOPC-21; BioLegend),isotype IgG2b PE (MPC-11; BioLegend), isotype IgG1 APC (MOPC-21; BDBioscience), isotype IgG2a APC (MOPC-173; BioLegend), isotype IgG2b APC(MG2b-57; BioLegend).

Western Blotting

Cells were lysed with RIPA buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl,0.1% SDS, 1% NP-40, 0.5% DOC) and boiled the extracts with Laemmlibuffer. Proteins were separated on 10% SDS-PAGE gels (Carl Roth) andtransferred to nitrocellulose membranes (GE Healthcare Life Science)using Mini-PROTEAN Tetra Cell (Bio-Rad). Membranes were blocked for 30minutes with Roti-Block (Carl Roth) followed by antibody incubation.Secondary antibodies conjugated with horseradish peroxidase were used(Cell Signaling) and exposed to CEA films (Agfa HealthCare). Proteinlevels were quantified with the software ImageJ. The following primaryantibodies reactive to human proteins were used: anti-human Tubulin(B-5-1-2; Santa Cruz), anti-human actin (AC-74; Sigma), anti-human IP07(ab88339; Abcam), anti-human TAP1 (1.28; Acris) and anti-human TAP2(2.17, Acris). The (TP-1467) monoclonal antibody reactive to the EBVprotein LMP2 was provided by Elisabeth Kremmer.

Luciferase Reporter Assays

The 3′-UTRs of IL12B (Ensembl: ENST00000231228) and TAP2 (Ensembl:ENST00000374897) were cloned downstream of firefly luciferase (Fluc) inthe expression plasmid psiCHECK-2 (Promega). To construct the viralmiRNA expression vectors, TagBFP (Evrogen) was clonedunder the controlof the EF1α promoter into pCDH-EF1-MCS (System Biosciences). SinglemiRNAs of interest were cloned downstream of the TagBFP-encoding gene.Viral miRNAs were obtained by PCR from the p4080 plasmid (Seto, loc.cit.). The psiCHECK-2 reporter and pCDH-EF1 miRNA expressor plasmid DNAswere co-transfected into HEK293T cells by Metafectene Pro (Biontex).After 24 hours of transfection, luciferase activities were measured withthe Dual-Luciferase Assay Kit (Promega) and the Orion II MicroplateLuminometer (Titertek-Berthold). The activity of Fluc was normalized tothe activity of Renilla luciferase (Rluc) encoded in the psiCHECK-2reporter. It was performed in silico prediction of EBV miRNA bindingsites on 3′-UTRs primarily with TargetScan (world wide webtargetscan.org) and employed RNAhybrid (world wide webbibiserv.techfak.uni-bielefeld.de/rnahybrid) to screen for 6mer bindingsites (Bartel, Cell. 136, 215-233 (2009)). Site-directed mutagenesiswere performed with overlapping oligo DNAs and Phusion polymerase (NEB).

Establishment of EBV-Stimulated Effector T-Cells and T-Cell Clones

EBV-specific CD8⁺ T-cell clones were established from polyclonal T-celllines that were generated by lymphoblastoid cell lines (LCLs) ormini-LCL stimulation of PBMCs as previously described (Adhikary et al.PLoS ONE. 2, e583 (2007))

T-Cell Differentiation and Recognition

Th1 differentiation was assessed by co-culture of sorted naive CD4⁺T-cells and infected B-cells 5 days post infection. 1×10⁵ naive CD4⁺T-cells stained with CellTrace Violet (Life Technologies) and 0.5 or1×10⁵ infected B-cells were cultured in 96 well plates with DynabeadsHuman T-Activator CD3/CD28 (Life Technologies) and cultivated for 7days. The neutralizing antibody against IL12B (C8.6; BioLegend) or thecorresponding isotype control antibody (MOPC-21; BioLegend) were addedfor certain experiments at 5 μg/ml. Cells were re-stimulated with PMAand ionomycin (Cell Stimulation Cocktail; eBioscience) for 5 hours andtreated with Brefeldin A and Monensin (Biolegend) for 2.5 hours prior tofixation. Th1 population was measured by intracellular IFN-y stainingwith FIX & PERM Cell Permeabilization Kit (Life Technologies) andsubsequent flow cytometery analysis. The Th1 population was defined asIFN-y positive T-cells in the fraction of proliferating T-cellsidentified via CellTrace Violet staining. EBV-specific effector T-cells'activities were measured with ELISA and Calcein release assays. ForIFN-γ detection from T-cells, effector and target cells were seeded at5×10⁴ cell per ml (1:1 ratio) each and co-cultured for 16 hours in a96-well plate (V bottom). IFN-γ levels were detected with ELISA. IFN-γconcentrations lower than 16 μg/ml were considered as not detected.

T-Cell Cytotoxicity Assays

Primary infected B-cells were purified by Ficoll-Hypaque gradientcentrifugation, and 5×10⁵ target cells were labeled with calcein at 0.5μg/ml. After three washing steps with PBS, target and effector cellswere co-cultured in a 96-well plate (V bottom) with different ratios inRPMI red phenol-free medium to reduce background signals. After fourhours of co-culture, fluorescence intensity of the released calcein wasmeasured by the Infinite F200 PRO fluorometer (Tecan). As controls,spontaneous calcein release of target cells cultivated without effectorcells and cells lysed with 0.5% Triton-X100 were used to define thelevels of no and fully lysed target cells, respectively.

Statistical Analysis

Prism 6.0 software (GraphPad) was used for the statistical analysis andtwo-tailed ratio T test was applied unless otherwise mentioned.

Example 1

Targets of EBV's miRNAs using an approach designed to detect cellularmRNAs the virus targets to foster its efficient infection were searched.Two stocks of EBV, a laboratory strain (wt/B95-8) that expresses 13miRNAs and its deleted derivative (ΔmiR) that expresses none, were usedto infect freshly isolated B-cells from six donors. RNAs were isolatedon day 5 following infection and sequenced. Genes that weredifferentially expressed were identified with those having a z-score>1.6shown in FIG. 1A. These genes included the viral miRNA targetsLY75/DEC205 (Skalsky et al., PLoS Pathog. 8, e1002484 (2012)) and IPO7(Dölken et al., Cell Host Microbe. 7, 324-334 (2010)). The identified,regulated genes were grouped according to the Kyoto Encyclopedia ofGenes and Genomes (KEGG) pathway categories (FIG. 5 ) based onconsistently down-regulated genes in wt/B95-8 EBV infected cells. Thisgrouping was enriched in the pathways linked to apoptosis, cell cycleregulation, and p53 signaling (Seto et al., PLoS Pathog. 6, e1001063(2010)). This grouping also strikingly revealed that in newly infectedcells, EBV's miRNAs regulate a wide array of immune functionsencompassing antigen processing, HLAs and co-stimulatory molecules, andcytokine-cytokine receptor interaction (FIG. 1B, FIG. 5 ). RNA-inducedsilencing complex (RISC-IP) was immunoprecipitated and found 14.5%(±2.4% SD) of all miRNAs were of viral origin in wt/B95-8 EBV-infectedcells (FIG. 10 and FIG. 6 ). It was also found that different mRNAs weredetected in the RISC differently among the cell samples as has beenfound in PAR-CLIP experiments (Skalsky, loc. cit.) (GEO: GSE41437).Therefore, the analyses were focused primarily on candidate mRNAsidentified by their differential expression in all samples (FIG. 1A) andused RISC-IP results to confirm them.

Example 2

It was confirmed that EBV's miRNAs regulate cytokines central to immunefunctions. The supernatants from B-cells infected with the two strainsof EBV were assayed for the levels of interleukin-6 (IL-6), IL-10,TNF-α, IL12B (IL-12p40), IL-12 (p35/p40), and IL-23 (p19/p40). CpG DNAwas added, which stimulates TLR9, for the detection of IL-6 and TNF-αsecreted from EBV-infected cells (Iskra, et al., J. Virol. 84, 3612-3623(2010)). The wt/B95-8 EBV-infected B-cells secreted less IL-6, TNF-α,and IL-12p40 than B-cells infected with ΔmiR EBV. In contrast, releaseof the anti-inflammatory cytokine IL-10 appeared to be unaffected byviral miRNAs (FIG. 2A) consistent with the transcriptome analysis (FIG.1B). Secretion of IL-12 (p35/p40) and IL-23 (p19/p40), both of whichcontain the IL-12p40 subunit (Szabo et al., Annu. Rev. Immunol. 21,713-758 (2003)), was significantly reduced in wt/B95-8 EBV-infectedcells compared with ΔmiR EBV-infected cells (FIG. 2A).

Example 3

It was found that EBV miRNAs directly regulate a cytokine-encoding geneIL12B, which encodes IL-12p40. The finding was verified with luciferasereporter assays. EBV's miR-BHRF1-2, miR-BART1, or miR-BART2 repressedthe luciferase activity of the IL12B reporter (FIG. 2B). The predictedbinding sites of miR-BART1 or miR-BART2 were mutated, which abrogatedtheir ability to inhibit the IL12B reporter (FIG. 2B and FIG. 7 )confirming the direct controls of viral miRNAs on this gene transcript.MiR-BART10 and miR-BART22 were analysed, which are present in fieldstrains of EBV but not in wt/B95-8 EBV, similarly (FIG. 2B and FIG. 7 ).Mutations of their predicted target sites only partially relieved theinhibition by both miRNAs, suggesting the presence of additional bindingsites for these miRNAs in the IL12B transcript. In summary, it wasconfirmed that cytokines are regulated by EBV miRNAs, and validatedIL128 as a direct target of multiple viral miRNAs.

Example 4

Additionally, levels of proteins pivotal to antigen processing andpresentation, including TAP1 and TAP2, whose transcript levels werereduced in wt/B95-8 compared with ΔmiR EBV-infected cells werequantified (FIG. 1B). Both TAP1 and TAP2 were decreased by EBV's miRNAs(FIG. 2C). They form a heterodimer, which mediates the cytoplasmictransport of antigenic peptides into the ER lumen, where they are loadedonto MHC class I molecules stabilizing them (Horst et al., J. Immunol.182,2313-2324 (2009)). MHC class I molecules and all three subclasses ofMHC class II molecules (HLA-DR, HLA-DQ and HLA-DP) were reduced as wereco-stimulatory and adhesion molecules by 15 days post infection (FIG. 2, D and E).

Example 5

RISC-IP and in silico algorithms indicated that the 3′-UTR of TAP2 istargeted by EBV miRNAs. In luciferase reporter assays miR-BHRF1-3repressed the TAP2 reporter (FIG. 2B). Mutations of the target motifabrogated repression of luciferase, indicating that TAP2 is a directtarget of miR-BHRF1-3 (FIG. 2B and FIG. 7 ). Similarly, miR-BART17,which is encoded by field strains of EBV, directly targeted the 3′-UTRof the TAP2 transcript (FIG. 2B and FIG. 7 ). Therefore, EBV miRNAsdown-regulate genes with pivotal functions in peptide antigenprocessing, transport and presentation early after infection.

Example 6

Viral miRNAs inhibit the secretion of IL-12 early after infection (FIGS.1B and 2A). This inhibition blocked differentiation of type 1 helper T(Th1) cells, a process for which IL-12 is critical (Szabo, loc. cit.).Naive CD4⁺ T-cells were co-cultured with autologous EBV-infected B-cells(FIG. 3A). The wt/B95-8 EBV-infected B-cells repressed Th1differentiation compared with ΔmiR EBV-infected cells (FIG. 3B). Anantibody that neutralizes the functions of IL12B, but not an isotypecontrol antibody, suppressed Th1 differentiation when the cells wereco-cultured with ΔmiR EBV-infected cells (FIG. 3C), indicating thatIL-12 secreted from EBV-infected or activated B-cells per se drives thegeneration of Th1 cells. Thus, EBV miRNAs suppress the release of IL-12from infected cells, a function that can abrogate antiviral control byvirus-specific Th1 cells.

Example 7

Further, inhibition of MHC class II, co-stimulatory, and adhesionmolecules by EBV miRNAs (FIGS. 1B and 2D, E) impaired MHC class Il-mediated recognition of infected cells by CD4⁺ T-cells. CD4⁺ T-cellswere expanded ex vivo by repeated stimulation with an irradiatedwt/B95-8 EBV-infected autologous lymphoblastoid cell line (LCL). TheEBV-specific CD4⁺ T-cells were then co-cultured with autologous B-cellsthat had been infected with the two EBV strains 5 days earlier (FIG.3D). Release of IFN-γ by EBV-specific CD4⁺ T-cells was substantial whenco-cultured with ΔmiR EBV-infected cells as targets but was consistentlyreduced when co-cultured with wt/B95-8 EBV-infected B-cells at all cellratios tested (FIG. 8 ). This effect was observed in autologous andHLA-matched but not in HLA-mismatched situations (FIG. 3E and FIG. 12 )indicating that the observed activation of CD4⁺ T-cells was HLA classII-restricted. An EBV antigen-specific CD4⁺ T-cell clone was testeddirected against the FGQ, an epitope from an EBV glycoprotein gp350(Adhikary, J. Exp. Med. 203, 995-1006 (2006)) and observed reducedT-cell activities with target B-cells infected with wt/B95-8 EBVcompared with ΔmiR EBVs five days after infection (FIG. 9 ). T-cellactivity against B-cells was barely detected at 15 days post infectionwhen the viral antigen gp350 was no longer present because it is acomponent of the virus particle and presented immediately after B-cellinfection (Adhikary, loc.cit.) but is not synthesized during latency(Kalla et al. Proc. Natl. Acad. Sci. U.S.A. 107, 850-855 (2010)).

EBV-specific CD4⁺ T-cells have cytolytic activity (Adhikary, loc. cit.).In allogeneic HLA-matched conditions, EBV-specific CD4⁺ T-cellsconsistently showed stronger cytolysis of target B-cells infected withΔmiR EBV than cells infected wt/B95-8 EBV (FIG. 3F). EBV miRNAs clearlyinhibited the recognition of infected B-cells by HLA class Il-restrictedCD4⁺ T-cells early after infection.

It was found also that EBV miRNAs impair recognition of infected B-cellsby MHC class I-restricted, EBV-specific CD8⁺ T-cells in addition to CD4⁺T-cells. These tests used co-culture assays with EBV-infected B-cellsand polyclonal EBV-specific CD8⁺ T-cells as well as CD8⁺ T-cell clonesspecific for certain EBV antigens. IFN-γ secretion by the CD8⁺ T-cellswas measured upon overnight cultivation with primary B-cells that hadbeen infected with the two different EBV strains 15 days earlier (FIG.4A). In accordance with their HLA restriction (and only in autologousand matched settings), CD8⁺ T-cells released IFN-γ after co-culture withΔmiR EBV-infected B-cells but less so when co-cultured with wt/B95-8EBV-infected B-cells (FIG. 4B and FIG. 12 ). Similarly, B-cells infectedwith ΔmiR EBV were significantly killed by EBV-specific CD8⁺ T-cellsrelative to B-cells infected with wt/B95-8 EBV expressing miRNAs (FIG.4C). Finally, IFN-γ release of the CD8⁺ T-cell clone specific for theIED epitope of viral protein LMP2 presented by HLA-B*40 (FIG. 10 )(Lautscham et al., J. Exp. Med. 194, 1053-1068 (2001)) was strongly andconsistently reduced when co-cultured with wt/B95-8 EBV-infected B-cellscompared with ΔmiR EBV-infected B-cells (FIG. 4D). HLA-B*40 but not LMP2expression was affected by EBV miRNAs (FIG. 4D and FIG. 11 ). Theseresults suggest that EBV miRNAs control antigen processing andpresentation to protect infected B-cells from the recognition byEBV-specific CD8⁺ T-cells.

Example 8

EBV eventually resides in most people in non-proliferating B-cellslargely invisible to a host's immune response (Thorley-Lawson, J.Allergy Clin. lmmunol. 116, 251-261 (2005)). However, it inducesproliferation of the B-cells it initially infects and fosters theirsurvival. It was found that EBV encodes miRNAs that regulate multiplefacets of a hosts adaptive immune response in newly infected B-cells.EBV-infected B-cells lacking viral miRNAs are deficient both inaffecting these responses and in other miRNA-dependent functionsincluding an inhibition of apoptosis (Seto, loc. cit.). These latterdefects have precluded comparisons of B-cells newly infected withwt/B95-8 or ΔmiR in humanized mouse models because of the defects insurvival of the latter cells (C. Münz, personal communication).Functional assays in culture show compellingly that EBV's miRNAs inhibitthe secretion of cytokines, inhibit antigen processing and presentation,inhibit the differentiation of CD4⁺ T-cells and their recognition ofinfected B-cells, and inhibit the recognition of those cells byEBV-specific CD8⁺ T-cells. The breadth of EBV's use of its miRNAs toinhibit adaptive and innate immune responses (Nachmani et al. Cell HostMicrobe. 5, 376-385 (2009)) is unprecedented and would foster itsefficient establishment of a life-long infection.

Example 9

VLP production was induced by transfection of producer cells asdescribed in Hettich et al. (Gene Therapy, 2006, vol. 13, pages844-856). The supernatant was filtered through a 1.2 μm filter andconcentrated by ultracentrifugation at 100,000×g for 2 hours. Finally,the pellet was resuspended in 1.5 mL PBS.

A human EBV-immortalized B-cell line (LCL) was plated into a 96-wellplate (5*10{circumflex over ( )}4 cells/well) and incubated with VLPs(1*10{circumflex over ( )}4 particles/cell) with miRNAs or lacking allmiRNAs (ΔmiRNAs) in a total volume of 200 μl/well. After 24 h ofincubation, 100 μl of the culture medium was removed and the cells werewashed by adding 100 μl of RPMI without supplements and centrifugationfor 5 minutes at 300×g. Again, 100 μl of the medium were removed andLCLs were mixed with an HLA-matched CD4+ T-cell clone (100 μl cellculture medium containing 5*10{circumflex over ( )}4 cells) specific forthe EBV tegument protein BNRF1 (ratio LCLs:T cells=1:1) and thenco-cultivated for another 24 hours. Activation of T cells was quantifiedin a IFNγ-ELISA assay according to the manufacturer's protocol (humanIFNγ-ELISA development kit (ALP), Mabtech). The assay was performed with5 technical replicates. Results of the assay are shown in FIG. 13 .

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used hereinthe terms “about” and “approximately” means within 10 to 15%, preferablywithin 5 to 10%. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof the invention are approximations, the numerical values set forth inthe specific examples are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in their respective testingmeasurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1. An Epstein-Barr VLP (EBVLP), comprising Epstein-Barr virus (EBV)proteins and lacking EBV miRNA, wherein said EBV miRNA is at least onebeing selected from the group consisting of miR-BART17, miR-BART10, andmiR-BART22.
 2. The EBVLP of claim 1, wherein said EBV miRNA is lackingdue to genetic modification to a nucleic acid molecule encoding theEBVLP, wherein the genetic modification effects that said EBV miRNA isnot expressed or only partially expressed, said EBV miRNA does not bindto its target sequence, said EBV miRNA or its precursor has a wrong 3Dstructure, the precursor of said EBV miRNA is not further processed,said EBV miRNA or its precursor are degraded by the cell, said EBV miRNAcoding loci has a scrambled sequence, said EBV miRNA coding loci isdeleted, and/or said EBV miRNA or its precursor comprises mutations,deletions or insertions.
 3. The EBVLP of claim 1, wherein said EBVLPleads to an increased immune response when compared to a EBVLP thatcomprises EBV miRNA, wherein said increase is at least 5% as determinedin a quantitative ELISA, comprising measuring the concentration ofproinflammatory cytokines in the supernatant of immune cells incubatedwith the EBVLP of claim 1 and comparing said cytokine concentration tothe cytokine concentration in the supernatant of immune cells incubatedwith EBVLPs comprising miRNA identical to the wild type virus.
 4. TheEBVLP of claim 1, wherein an at least one nucleic acid molecule encodingsaid EBV proteins is genetically modified such that it is not packagedin the EBVLPs.
 5. The EBVLP of claim 1, wherein said EBVLP issubstantially free of an EBV genome and/or an at least one nucleic acidmolecule encoding the EBV proteins.
 6. The EBVLP of claim 1, wherein atleast one nucleic acid molecule encoding said EBV proteins comprises (i)at least one gene encoding an EBV protein required for cellulartransformation, which is genetically modified such that said EBV proteinis not expressed or non-functional; and/or (ii) at least one geneencoding an EBV protein required for inducing virus synthesis, which isgenetically modified such that said EBV protein is not expressed ornon-functional.
 7. The EBVLP of claim 1, wherein (i) an at least onenucleic acid molecule encoding said EBV proteins comprises at least onegene, encoding an EBV protein required for B-cell transformation,selected from the group consisting of EBNA1, EBNA-LP, EBNA2, LMP1, LMP2,EBNA3A, and EBNA3C, which is genetically modified such that the EBVprotein is not expressed or non-functional; (ii) an at least one nucleicacid molecule encoding said EBV proteins comprises at least one gene,encoding an EBV protein required for inducing virus synthesis, selectedfrom the group consisting of BZFL1, BRLF1 and BMLF1, which isgenetically modified such that the EBV protein is not expressed ornon-functional; (iii) an at least one nucleic acid molecule encodingsaid EBV proteins lacks the packaging element TR; (iv) an at least onenucleic acid molecule encoding said EBV proteins comprises at least onegene encoding an EBV protein required for packaging of EBV DNA, selectedfrom the group consisting of BFLF1, BBRF1, BGRF1, BDRF1, BALF3, BFRF1A,and BFRF1, which is genetically modified such that said EBV protein isnot expressed or non-functional; and/or (v) an at least one nucleic acidmolecule encoding said EBV proteins comprises an EBV genome.
 8. Anucleic acid molecule encoding the EBV proteins of the EBVLP of claim 1.9. A vector comprising the nucleic acid molecule of claim
 8. 10. Acomposition of matter comprising at least two nucleic acid moleculesencoding the EBV proteins of the EBVLP of claim
 1. 11. An isolated hostcell transfected with a nucleic acid molecule encoding the EBV proteinsof the EBVLP of claim 1, a vector comprising said nucleic acid encodingthe EBV proteins of the EBVLP or a composition comprising at least twonucleic acid molecules encoding the EBV proteins of the EBVLP ofclaim
 1. 12. A method for generating an Epstein-Barr VLP (EBVLP), themethod comprising: (i) culturing the isolated host cell of claim 11under conditions that allow expression of the EBV proteins; and (ii)obtaining said EBVLP.
 13. The method of claim 12, comprising after step(i) and prior to step (ii) a further step (i′), comprising inducing thereplicative phase of the Epstein-Barr virus, wherein said replicativephase is induced by expressing at least one gene, encoding an EBVprotein that is required for inducing EBV synthesis, wherein the atleast one gene, comprised by the at least one nucleic acid moleculeencoding the EBV proteins of the EBVLP, has been genetically modified,such that said EBV protein is not expressed or non-functional.
 14. Avaccine composition comprising the EBVLP of claim 1 further comprisingan excipient.
 15. The vaccine composition of claim 14, furthercomprising one or more viral or non-viral polypeptides, one or moreviral or non-viral nucleic acid sequences and/or vaccine adjuvants,wherein said one or more viral polypeptides or said one or more viralnucleic acid sequences are not from the same virus as the EBVLP in saidvaccine composition.
 16. A method of treating or preventing a diseaseassociated with EBV infection in a subject comprising administering theEBVLP of claim 1, or a vaccine composition comprising the EBVLP of claim1, and/or a vaccine composition comprising the EBVLP of claim 1 and anexcipient, and further comprising one or more viral or non-viralpolypeptides, one or more viral or non-viral nucleic acid sequencesand/or vaccine adjuvants, wherein said one or more viral polypeptides orsaid one or more viral nucleic acid sequences are not from the samevirus as the EBVLP in said vaccine composition, for vaccination ortreatment of the subject.
 17. A kit comprising the EBVLP of claim 1, anucleic acid molecule encoding the EBV proteins of the EBVLP of claim 1,a vector comprising said nucleic acid encoding the EBV proteins of theEBVLP of claim 1, a composition comprising at least two nucleic acidmolecules encoding the EBV proteins of the EBVLP of claim 1, an isolatedhost cell comprising said nucleic acid molecule, a vaccine compositioncomprising the EBVLP of claim 1, and/or a vaccine composition comprisingthe EBVLP of claim 1 and an excipient, and further comprising one ormore viral or non-viral polypeptides, one or more viral or non-viralnucleic acid sequences and/or vaccine adjuvants, wherein said one ormore viral polypeptides or said one or more viral nucleic acid sequencesare not from the same virus as the EBVLP in said vaccine composition.18. The method of claim 12, wherein a genetic modification to thenucleic acid molecule, which the isolated host cell is transfected with,encoding the EBVLP effects that EBV miRNA is not expressed or onlypartially expressed, EBV miRNA does not bind to its target sequence, EBVmiRNA or its precursor has a wrong 3D structure, the precursor of EBVmiRNA is not further processed, EBV miRNA or its precursor are degradedby the cell, EBV miRNA coding loci has a scrambled sequence, EBV miRNAcoding loci is deleted, and/or EBV miRNA or its precursor comprisesmutations, deletions or insertions.
 19. The EBVLP of claim 4, wherein(i) the at least one nucleic acid molecule encoding said EBV proteinslacks a functional cis-acting element required for packaging; or (ii)the at least one nucleic acid molecule encoding said EBV proteinscomprises at least one gene encoding an EBV protein required forpackaging, which is genetically modified such that said EBV protein isnot expressed or non-functional.
 20. The method of claim 13, wherein (a)said gene is expressed from a stably transfected vector comprised bysaid host cell and/or wherein expression of said gene is induciblyregulated, and/or (b) said gene encoding said EBV protein is selectedfrom the group consisting of BZLF1, BRLF1 and BMLF1.